HUMAN IMMUNOLOGY doi: 10.1111/sji.12202 ..................................................................................................................................................................
Autoantibodies Response to MDM2 and p53 in the Immunodiagnosis of Esophageal Squamous Cell Carcinoma Y. Chai*†, B. Peng†, L. Dai*†, W. Qian†, Y. Zhang* & J.-Y. Zhang†
Abstract *Center for Tumor Biotherapy, The First Affiliated Hospital, College of Public Health, Zhengzhou University, Zhengzhou, Henan 450052, China; and †Department of Biological Sciences, The University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968, USA
Received 28 March 2014; Accepted in revised form 5 June 2014 Correspondence to: Dr J.-Y. Zhang, Department of Biological Sciences, The University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968, USA. E-mail: [email protected]
The human homologue of the mouse double minute 2 (MDM2) is known to be overexpressed in a variety of human malignancies. As one of E3 ubiquitin– protein ligases, MDM2 interacts with the tumour suppressor p53 by mediating ubiquitination and degradation of p53. Since abnormally expressed proteins can induce autoimmune response, to further examine whether sera from patients with esophageal squamous cell carcinoma (ESCC) exhibited immunoreactivity against MDM2 and p53, autoantibody responses to MDM2 and p53 were evaluated by enzyme-linked immunosorbent assay (ELISA) in sera from patients with ESCC and normal individuals. Positive results were also confirmed by Western blotting and indirect immunofluorescence assay. The results demonstrated that the positive rate of autoantibody against p53 and MDM2 in ESCC sera was 22.9% (36/157) and 14.0% (22/157), whereas this rate was 0% (0/85) and 1.2% (1/85), respectively, in normal individuals. Some of the sera with antibodies specific for MDM2 also contained antibodies against p53. And there was an increase of positive antibody reactions reaching a frequency of 35% (55/157) combination with MDM2 and p53. This was significantly higher than the frequency of antibodies in normal individuals (P < 0.01). Our preliminary results suggest that autoantibodies against MDM2 and p53 may be useful serum biomarkers in the immunodiagnosis of ESCC.
Introduction Cancer has long been recognized as a highly complex disorder that involves not only the interplay of genomic changes but also factors including the immunological, hormonal, environmental and other disease [1]. It has demonstrated that cancer sera contain antibodies that react with a unique group of autologous cellular antigens called tumour-associated antigens (TAAs) [2, 3]. These antigens are probably cellular proteins, such as p53, p62, p90 and p16, whose aberrant regulation or overexpression is capable of leading to tumourigenesis [4–8]. The cancer-associated autoantibodies may be regarded as ‘reporters’ identifying aberrant or disregulated cellular mechanisms involved in the progress of transformation [9]. Esophageal squamous cell carcinoma (ESCC) has become one of six prevalent malignancies in China [10]. Due to the fact that the early-stage ESCC has no obvious symptom and there is no acceptable diagnostic method for early detection, more than 95% ESCC patients from rural regions were diagnosed at middle and advanced stages [11].
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Therefore, identification of effective sera molecular biomarkers with less invasive is crucially vital for early detection and screening for ESCC. p53, as a tumour suppressor protein, is one of the most extensively studied TAAs. Autoantibodies to p53 in sera from patients with breast cancer were first reported in 1982 [12]. Autoantibodies to p53 have been detected in some cases before cancer diagnosis [13]. Since then, there have been numerous reports confirming and extending this finding that sera from cancer patients frequently contain antibodies specific for p53, including hepatocellular carcinoma and ESCC [14, 15]. The human homologue of the mouse double minute 2 (MDM2), also known as E3 ubiquitin–protein ligase, has been shown to be overexpressed in a variety of human neoplasms. MDM2 overexpression significantly enhanced cell invasion, migration in invasive ductal breast cancer (IDC) [16]. It was also reported that MDM2 overexpression might be responsible for the accumulated p53 value in IDC and the accumulated p53 was significantly associated with MDM2-positive cells [17]. MDM2 can inhibit p53 accumulation by targeting it
Ó 2014 John Wiley & Sons Ltd
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for ubiquitination and proteasomal degradation [18]. Moreover, DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2 [19]. According to these observations, we wonder whether the sera from patients with ESCC may develop autoantibodies against interacting partner molecules of MDM2 and p53. To understand the immunogenicity of them, in the current study, autoantibodies to MDM2 and p53 were evaluated and validated to be potential biomarkers in immunodiagnosis of ESCC.
Materials and methods Sera and general information. In the current study, a total of 157 sera from patients with ESCC and 85 normal human sera (NHS) was obtained from the serum bank of Cancer Autoimmunity and Epidemiology Research Laboratory at University of Texas, El Paso (UTEP). The sera were originally provided by our clinical collaborators. All patients were confirmed by histopathological diagnosis. Cancer sera were collected at the time of initial cancer diagnosis, when the patients had not yet received treatment with any chemotherapy or radiation therapy. Of 157 ESCC patients, 115 (73.2%) were males, and 42 (26.8%) were females. Mean age was 59.0 11.8 years (range, 26– 78 years). NHS were collected during annual health examinations from people who had no obvious evidence of malignancy from the same locality. Due to regulations concerning studies of human subjects, the subjects’ name and identification number were blinded to investigators, and some clinical information for sera used in this study was not currently available. This study was approved by the Institutional Review Board of UTEP and collaborating institutions. Expression and purification of recombinant MDM2 and p53. Recombinant proteins of MDM2 and p53 were derived from our previous studies [14]. p53 and MDM2 cDNAs were respectively subcloned into pET28a vector producing fusion proteins with NH-terminal 69 histidine and T7 epitope tags. Recombinant proteins were further expressed in E. coli BL21 (DE3) and purified using nickel column chromatography (Qiagen, Valencia, CA, USA). Reactivities of the purified recombinant proteins were further analyzed by electrophoresis on SDS-PAGE and determined with either polyclonal anti-MDM2 antibody (GeneTex, Irvine, CA, USA) or monoclonal antibody against p53 (Cell Signaling, Danvers, CO, USA) as well as the ESCC serum antibodies which were used for immunoscreening. Enzyme-linked immunosorbent assay (ELISA). Standard protocol for ELISA was used as described in our previous study [20]. In brief, a 96-well microtitre plate was coated overnight at 4 °C with recombinant MDM2 or p53 proteins at a final concentration of 0.5 lg/ml in phosphate-buffered Ó 2014 John Wiley & Sons Ltd
saline (PBS). The antigen-coated wells were blocked with gelatin post-coating solution at room temperature for 2 h. Human sera diluted at 1:100 with serum diluent were incubated for 2 h at room temperature in the antigen-coated wells, followed by HRP-conjugated goat anti-human IgG. The substrate 2,20 -azino-bis-3-ethylbenzo-thiazoline-6-sulphonic acid (ABTS; Sigma-Aldrich, St. Louis, MO, USA) was used as detecting reagent. The average optical density (OD) value at a wavelength of 405 nm was applied as data analysis. The cut-off value designating a positive reaction was the mean OD of 85 NHS + 3SD. Western blotting. Denatured recombinant MDM2 and p53 proteins were electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membranes, respectively. After blocking in PBS with 5% non-fat milk and 0.05% Tween20 for 1 h at room temperature, the nitrocellulose membrane were incubated overnight at 4 °C with 1:200 dilution of human sera, 1:1000 dilution of polyclonal antiMDM2 antibody or monoclonal antibody against p53, separately. HRP-conjugated goat anti-human IgG, goat anti-rabbit IgG and goat anti-mouse IgG (Santa Cruz, CA, USA) were applied as secondary antibody at a 1:3000 dilution, respectively. The ECL kit was used to detect immunoreactive bands according to the manufacturer’s instructions (Thermo Scientific, Waltham, MA, USA). Absorption of antibodies with recombinant protein. The diluted human ESCC sera (1:80) were incubated with recombinant proteins MDM2 and p53 (final concentration of recombinant proteins in the diluted human sera was 0.01 lg/ll) overnight at 4 °C, and then centrifuged at 10,000 g for 15 min. The supernatant was used for immunofluorescence assay. Indirect immunofluorescence assay. Hep-2 antigen substrate for Indirect immunofluorescence assay (IIFA) test system was incubated with dilution of sera (1:80) and pre-absorbed sera overnight at 4 °C. FITC-conjugated goat anti-human IgG was used as secondary antibody at a 1:100 dilution. Fluorescence microscope (Leica DM1000; Wetzlar, Germany) was used for examination. Statistical analysis. All data are represented as means standard deviation (SD). The frequency of autoantibodies to MDM2 and p53 in the sera was compared using the v2 test with Fisher’s exact test. Statistical analysis was performed in SPSS 13.0 software (Chicago, Illinois, USA). P < 0.01 was considered as statistically significant.
Results Frequency and titre of autoantibody against MDM2 and p53 in sera from patients with ESCC
Serum levels of anti-MDM2 and anti-p53 autoantibodies were determined by ELISA as described in the section of Materials and Methods. Totally, 157 sera from patients
364 Autoantibodies to MDM2 and p53 in Esophageal Squamous Cell Carcinoma Y. Chai et al. .................................................................................................................................................................. with ESCC and 85 sera from normal human individuals were used in this study. As shown in Table 1, higher frequency (P < 0.01) of antibodies with individual MDM2 and p53 in ESCC was found at 14.0% (22/157) in MDM2 and at 22.9 (36/157) in p53. The reactivity of NHS was low, the frequencies were 0% (0/85, against p53) and 1.2% (1/85, against MDM2), respectively. Table 1 also showed the comprehensive evaluation of antibodies of MDM2 and p53. In the combinational panel of MDM2 and p53, there was an increase of positive antibody reactions up to 35% (55/157). It suggests that parallel assay of TAAs can raise the diagnostic precision greatly. Titre of anti-MDM2 and anti-p53 antibodies in sera from patients with ESCC and normal individuals was shown in Fig. 1. The average titre of autoantibody against MDM2 and p53 in ESCC sera was higher than that in NHS (P < 0.01). Many ESCC sera showed OD values several fold above the cutoff (mean + 3SD of normal sera). Positive ELISA results were also confirmed by Western blotting analysis. Eight representative ESCC sera which were positive in ELISA had strong reactivity with MDM2 recombinant protein in Western blotting analysis. The same sera also had reactivity with p53 recombinant protein in Western blotting except the serum in lane 8 (Fig. 2). It Table 1 Frequency of autoantibody against MDM2 and p53 in human sera by ELISA. Type of sera ESCC NHS
Number
Autoantibody to MDM2 (%)
Autoantibody to p53 (%)
MDM2 or p53 (%)
157 85
22 (14.0)** 1 (1.2)
36 (22.9)** 0 (0.0)
55 (35)** 1 (1.2)
ESCC, esophageal squamous cell carcinoma; NHS, normal human sera; MDM2, mouse double minute 2. **P < 0.01 (ESCC versus NHS).
Figure 1 Titer of autoantibodies against MDM2 and p53 in esophageal squamous cell carcinoma (ESCC) patients and normal human sera (NHS). The range of antibody titers to MDM2 and p53 is expressed as optical density (OD) obtained from ELISA. The Y-axis represents the OD value. The X-axis represents serum samples. Titer of anti-MDM2 and p53 in ESCC was much higher than that in NHS (P < 0.01).
seemed that there were higher positive rate with MDM2 or p53 antibody reactivity in Western blotting compared with that in ELISA assay. For the possibilities that related to recognition of different epitopes on the same antigen in the ELISA assay and Western blotting, or the different sensitivity between two assays, the results of the two immunological assays varied. Immunofluorescence staining pattern of MDM2 and p53 in Hep-2 cells
To further confirm the reactivity of autoantibodies against MDM2 and p53 in ESCC sera and the intracellular localization of MDM2 and p53, Hep-2 cell slides were used in IIFA to detect ESCC sera with anti-MDM2 and anti-p53 positive in ELISA. As shown in Fig. 3, an anti-MDM2 positive ESCC serum had both cytoplasmic and nuclear staining pattern, while the polyclonal anti-MDM2 antibody with more intense staining in perinuclear regions. The normal human serum had very weak stain. The same serum with anti-p53 had little cytoplasmic and more nuclear staining pattern, while the monoclonal anti-p53 antibody with only intense staining in nuclear regions (Fig. 4). The fluorescent staining was significantly reduced when the same ESCC serum was pre-absorbed with recombinant anti-MDM2 or anti-p53 protein.
Discussion The possible mechanisms underlying the formation of autoantibodies and the regulation of their production remain unknown. Some reports have suggested that this immune response is likely to be related to the dysregulation of the function of these proteins or change of their molecular structure or location during malignant transformation [21]. It was demonstrated that the enhanced B cell toll like receptor 7 expression permitted the specific development of antibodies to RNA/protein complexes and drived anti-RNA autoantibody production [22]. One of the most extensively studied TAAs is p53, which is crucial for cellular stress response and tumour suppression pathways. Sera from cancer patients frequently contain antibodies
Figure 2 Western blotting analysis with representative sera in ELISA. Lane (1) The polyclonal anti-MDM2 antibody and monoclonal anti-p53 antibody were used as positive controls. Lane (2–9) eight representative ESCC sera which were positive in ELISA also had strong reactivity with MDM2 recombinant protein in western blotting analysis. The same sera also have reactivity with p53 recombinant protein except the serum in lane (8).
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A
B
C
Figure 3 Immunofluorescence staining pattern of anti-MDM2 antibody positive in esophageal squamous cell carcinoma (ESCC) serum. (A) A normal human serum (NHS) was used as a negative control. (B) The polyclonal anti-MDM2 antibody that demonstrated both cytoplasmic and perinuclear immunofluorescence staining pattern. (C) A anti-MDM2 antibody positive ESCC serum immunofluorescence staining pattern. (D) The same ESCC serum which used in panel C was pre-absorbed with recombinant MDM2 protein. The fluorescent signal was remarkably decreased.
D
specific for p53 [4, 23]. A study showed that lung tumours containing several types of p53 gene mutations, including missense, stop codon, splice/stop, splice, or frameshift mutations, only the p53 missense mutations, with overexpression of a protein that altered function and increased protein stability, correlated with p53 autoantibody production [24]. It was also demonstrated that there was a statistically significant relationship between p53 autoantibody and the subsequent development of malignancy, and therefore p53 autoantibody can have high predictive value for cancer [13]. Some studies further demonstrated that serum anti-p53 antibodies could be used as early markers for malignant tumours or markers for poor prognosis [25, 26]. p53 plays an important role in cellular physiological and pathological process. It can also form complexes with the MDM2 protein which results in stabilization of p53 [27, 28]. Some patients with antibodies against MDM2 also developed antibodies against p53 [29]. In the sera of Ó 2014 John Wiley & Sons Ltd
ESCC patients autoantibodies against p53 has been detected [30]. Because of the important roles of MDM2 and p53 in cancer formation, we try to validate whether anti-MDM2 and anti-p53 antibodies can be used as biomarkers for early ESCC diagnosis in this study. To test the prevalence of anti-MDM2 and anti-p53 autoantibodies in ESCC patients, recombinant proteins MDM2 and p53 were used as coating antigens in ELISA to detect autoantibodies against the antigens in 157 sera from patients with ESCC and 85 sera from normal individuals. Antibody frequency to individual MDM2 and p53 in ESCC was 14% and 22.9%, respectively. It cannot meet the needs of clinical early diagnosis to ESCC. Both anti-MDM2 and anti-p53 were simultaneously used as diagnostic markers, 35% ESCC cancer patients could be correctly identified, which showed that the clinical diagnostic value of parallel assay of TAAs was high. These data showed the utility of the
366 Autoantibodies to MDM2 and p53 in Esophageal Squamous Cell Carcinoma Y. Chai et al. .................................................................................................................................................................. A
B
C
multiple antigen array in increasing the clinical diagnostic quality and value in cancer. p53 is one of the most frequently mutated genes in cancer. There are multiple ways by which p53 can be regulated. MDM2 is the major regulator of p53 that targets p53 for proteasomal degradation [31, 32]. MDM2 interacts with the transactivation domain of p53 through its N-terminus. MDM2 not only degrades p53 but also regulates its localization. Overexpression of MDM2 is found in a wide variety of human tumours, for example, oral squamous cell carcinomas, breast carcinoma, sarcomas, and leukaemia, due to one of three different mechanisms: gene amplification, increased transcription or enhanced translation [33, 34]. In ovarian carcinomas, 48% of cases showed MDM2 gene amplification [35] However, in ESCC tissues the overexpression of MDM2 was not significantly associated with the abnormal expression of p53 [36]. In this study, we found that some ESCC sera which had strong reactivity with MDM2 recombinant protein also had reactivity with p53 recombinant protein in Western blotting analysis, which indicated that recombinant proteins of MDM2 and p53 as antigens had been shown to be capable of inducing autoantibodies in patients with ESCC. These results were also confirmed by IIFA and it was found that there were strong cytoplasmic and perinuclear expressions of MDM2 and p53 in sera from patients with
Figure 4 Immunofluorescence staining pattern of anti-p53 antibody positive in ESCC serum. (A) Monoclonal anti-p53 antibody that demonstrated a nuclear immunofluorescence staining pattern was used as positive control. (B) A anti-p53 antibody positive ESCC serum mainly nuclear immunofluorescence staining pattern. (C) The same ESCC serum which used in panel b was pre-absorbed with recombinant p53 protein. The fluorescent signal of nuclear immunofluorescence was remarkably decreased.
ESCC. These results indicated that autoantibodies against MDM2 and p53 in ESCC sera might be potential biomarkers for early-stage ESCC screening and diagnosis. Unlike traditional tumour biomarkers, anti-TAA autoantibodies are found in patients’ sera with different cancers, and may represent early indicators of tumour development [37]. The molecular characterization and identification of TAAs in cancers will also contribute to understanding of their functions in malignant transformation, thereby providing attractive candidates for early diagnosis and targeted therapies. Though MDM2 and p53 were overexpressed in many types of cancer tissues and it was demonstrated that MDM2 protein caused the high proliferative activity of ameloblastoma [38], measurement of serum anti-MDM2 antibody was not so much conducted like serum anti-p53 antibody. It was also found that serum anti-p53 antibody measurement does not contribute to the diagnosis of pancreatobiliary cancers [39]. Therefore, identification of novel serum biomarkers for the diagnosis of cancer, especially for detection and screening in earlystage cancer, is an important means to improve clinical outcome [40]. In conclusion, a total of 157 sera from patients with ESCC have been tested with different immunological assay. The data in our study suggests that MDM2 and p53 can induce autoantibody responses in sera from patients with
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ESCC, indicating that anti-MDM2 and anti-p53 autoantibodies might be potential biomarkers for clinical serologic screening in the diagnosis of ESCC. Further studies to elucidate the mechanism of anti-MDM2 and anti-p53 autoantibodies production will be necessary for a better understanding of the immune status in ESCC.
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Acknowledgment
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The authors thank Dr. Eng M. Tan (The Scripps Research Institute, USA) for his support. This work was supported by grants from the National Natural Science Foundation of China (81372371), and also by a grant (SC1CA166016) from the National Institutes of Health of USA (NIH).
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Conflict of interest
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The authors declare that there is no conflict of interests regarding the publication of this paper.
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Autoantibodies Response to MDM2 and p53 in the Immunodiagnosis of Esophageal Squamous Cell Carcinoma Y. Chai*†, B. Peng†, L. Dai*†, W. Qian†, Y. Zhang* & J.-Y. Zhang†
Abstract *Center for Tumor Biotherapy, The First Affiliated Hospital, College of Public Health, Zhengzhou University, Zhengzhou, Henan 450052, China; and †Department of Biological Sciences, The University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968, USA
Received 28 March 2014; Accepted in revised form 5 June 2014 Correspondence to: Dr J.-Y. Zhang, Department of Biological Sciences, The University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968, USA. E-mail: [email protected]
The human homologue of the mouse double minute 2 (MDM2) is known to be overexpressed in a variety of human malignancies. As one of E3 ubiquitin– protein ligases, MDM2 interacts with the tumour suppressor p53 by mediating ubiquitination and degradation of p53. Since abnormally expressed proteins can induce autoimmune response, to further examine whether sera from patients with esophageal squamous cell carcinoma (ESCC) exhibited immunoreactivity against MDM2 and p53, autoantibody responses to MDM2 and p53 were evaluated by enzyme-linked immunosorbent assay (ELISA) in sera from patients with ESCC and normal individuals. Positive results were also confirmed by Western blotting and indirect immunofluorescence assay. The results demonstrated that the positive rate of autoantibody against p53 and MDM2 in ESCC sera was 22.9% (36/157) and 14.0% (22/157), whereas this rate was 0% (0/85) and 1.2% (1/85), respectively, in normal individuals. Some of the sera with antibodies specific for MDM2 also contained antibodies against p53. And there was an increase of positive antibody reactions reaching a frequency of 35% (55/157) combination with MDM2 and p53. This was significantly higher than the frequency of antibodies in normal individuals (P < 0.01). Our preliminary results suggest that autoantibodies against MDM2 and p53 may be useful serum biomarkers in the immunodiagnosis of ESCC.
Introduction Cancer has long been recognized as a highly complex disorder that involves not only the interplay of genomic changes but also factors including the immunological, hormonal, environmental and other disease [1]. It has demonstrated that cancer sera contain antibodies that react with a unique group of autologous cellular antigens called tumour-associated antigens (TAAs) [2, 3]. These antigens are probably cellular proteins, such as p53, p62, p90 and p16, whose aberrant regulation or overexpression is capable of leading to tumourigenesis [4–8]. The cancer-associated autoantibodies may be regarded as ‘reporters’ identifying aberrant or disregulated cellular mechanisms involved in the progress of transformation [9]. Esophageal squamous cell carcinoma (ESCC) has become one of six prevalent malignancies in China [10]. Due to the fact that the early-stage ESCC has no obvious symptom and there is no acceptable diagnostic method for early detection, more than 95% ESCC patients from rural regions were diagnosed at middle and advanced stages [11].
362
Therefore, identification of effective sera molecular biomarkers with less invasive is crucially vital for early detection and screening for ESCC. p53, as a tumour suppressor protein, is one of the most extensively studied TAAs. Autoantibodies to p53 in sera from patients with breast cancer were first reported in 1982 [12]. Autoantibodies to p53 have been detected in some cases before cancer diagnosis [13]. Since then, there have been numerous reports confirming and extending this finding that sera from cancer patients frequently contain antibodies specific for p53, including hepatocellular carcinoma and ESCC [14, 15]. The human homologue of the mouse double minute 2 (MDM2), also known as E3 ubiquitin–protein ligase, has been shown to be overexpressed in a variety of human neoplasms. MDM2 overexpression significantly enhanced cell invasion, migration in invasive ductal breast cancer (IDC) [16]. It was also reported that MDM2 overexpression might be responsible for the accumulated p53 value in IDC and the accumulated p53 was significantly associated with MDM2-positive cells [17]. MDM2 can inhibit p53 accumulation by targeting it
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for ubiquitination and proteasomal degradation [18]. Moreover, DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2 [19]. According to these observations, we wonder whether the sera from patients with ESCC may develop autoantibodies against interacting partner molecules of MDM2 and p53. To understand the immunogenicity of them, in the current study, autoantibodies to MDM2 and p53 were evaluated and validated to be potential biomarkers in immunodiagnosis of ESCC.
Materials and methods Sera and general information. In the current study, a total of 157 sera from patients with ESCC and 85 normal human sera (NHS) was obtained from the serum bank of Cancer Autoimmunity and Epidemiology Research Laboratory at University of Texas, El Paso (UTEP). The sera were originally provided by our clinical collaborators. All patients were confirmed by histopathological diagnosis. Cancer sera were collected at the time of initial cancer diagnosis, when the patients had not yet received treatment with any chemotherapy or radiation therapy. Of 157 ESCC patients, 115 (73.2%) were males, and 42 (26.8%) were females. Mean age was 59.0 11.8 years (range, 26– 78 years). NHS were collected during annual health examinations from people who had no obvious evidence of malignancy from the same locality. Due to regulations concerning studies of human subjects, the subjects’ name and identification number were blinded to investigators, and some clinical information for sera used in this study was not currently available. This study was approved by the Institutional Review Board of UTEP and collaborating institutions. Expression and purification of recombinant MDM2 and p53. Recombinant proteins of MDM2 and p53 were derived from our previous studies [14]. p53 and MDM2 cDNAs were respectively subcloned into pET28a vector producing fusion proteins with NH-terminal 69 histidine and T7 epitope tags. Recombinant proteins were further expressed in E. coli BL21 (DE3) and purified using nickel column chromatography (Qiagen, Valencia, CA, USA). Reactivities of the purified recombinant proteins were further analyzed by electrophoresis on SDS-PAGE and determined with either polyclonal anti-MDM2 antibody (GeneTex, Irvine, CA, USA) or monoclonal antibody against p53 (Cell Signaling, Danvers, CO, USA) as well as the ESCC serum antibodies which were used for immunoscreening. Enzyme-linked immunosorbent assay (ELISA). Standard protocol for ELISA was used as described in our previous study [20]. In brief, a 96-well microtitre plate was coated overnight at 4 °C with recombinant MDM2 or p53 proteins at a final concentration of 0.5 lg/ml in phosphate-buffered Ó 2014 John Wiley & Sons Ltd
saline (PBS). The antigen-coated wells were blocked with gelatin post-coating solution at room temperature for 2 h. Human sera diluted at 1:100 with serum diluent were incubated for 2 h at room temperature in the antigen-coated wells, followed by HRP-conjugated goat anti-human IgG. The substrate 2,20 -azino-bis-3-ethylbenzo-thiazoline-6-sulphonic acid (ABTS; Sigma-Aldrich, St. Louis, MO, USA) was used as detecting reagent. The average optical density (OD) value at a wavelength of 405 nm was applied as data analysis. The cut-off value designating a positive reaction was the mean OD of 85 NHS + 3SD. Western blotting. Denatured recombinant MDM2 and p53 proteins were electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membranes, respectively. After blocking in PBS with 5% non-fat milk and 0.05% Tween20 for 1 h at room temperature, the nitrocellulose membrane were incubated overnight at 4 °C with 1:200 dilution of human sera, 1:1000 dilution of polyclonal antiMDM2 antibody or monoclonal antibody against p53, separately. HRP-conjugated goat anti-human IgG, goat anti-rabbit IgG and goat anti-mouse IgG (Santa Cruz, CA, USA) were applied as secondary antibody at a 1:3000 dilution, respectively. The ECL kit was used to detect immunoreactive bands according to the manufacturer’s instructions (Thermo Scientific, Waltham, MA, USA). Absorption of antibodies with recombinant protein. The diluted human ESCC sera (1:80) were incubated with recombinant proteins MDM2 and p53 (final concentration of recombinant proteins in the diluted human sera was 0.01 lg/ll) overnight at 4 °C, and then centrifuged at 10,000 g for 15 min. The supernatant was used for immunofluorescence assay. Indirect immunofluorescence assay. Hep-2 antigen substrate for Indirect immunofluorescence assay (IIFA) test system was incubated with dilution of sera (1:80) and pre-absorbed sera overnight at 4 °C. FITC-conjugated goat anti-human IgG was used as secondary antibody at a 1:100 dilution. Fluorescence microscope (Leica DM1000; Wetzlar, Germany) was used for examination. Statistical analysis. All data are represented as means standard deviation (SD). The frequency of autoantibodies to MDM2 and p53 in the sera was compared using the v2 test with Fisher’s exact test. Statistical analysis was performed in SPSS 13.0 software (Chicago, Illinois, USA). P < 0.01 was considered as statistically significant.
Results Frequency and titre of autoantibody against MDM2 and p53 in sera from patients with ESCC
Serum levels of anti-MDM2 and anti-p53 autoantibodies were determined by ELISA as described in the section of Materials and Methods. Totally, 157 sera from patients
364 Autoantibodies to MDM2 and p53 in Esophageal Squamous Cell Carcinoma Y. Chai et al. .................................................................................................................................................................. with ESCC and 85 sera from normal human individuals were used in this study. As shown in Table 1, higher frequency (P < 0.01) of antibodies with individual MDM2 and p53 in ESCC was found at 14.0% (22/157) in MDM2 and at 22.9 (36/157) in p53. The reactivity of NHS was low, the frequencies were 0% (0/85, against p53) and 1.2% (1/85, against MDM2), respectively. Table 1 also showed the comprehensive evaluation of antibodies of MDM2 and p53. In the combinational panel of MDM2 and p53, there was an increase of positive antibody reactions up to 35% (55/157). It suggests that parallel assay of TAAs can raise the diagnostic precision greatly. Titre of anti-MDM2 and anti-p53 antibodies in sera from patients with ESCC and normal individuals was shown in Fig. 1. The average titre of autoantibody against MDM2 and p53 in ESCC sera was higher than that in NHS (P < 0.01). Many ESCC sera showed OD values several fold above the cutoff (mean + 3SD of normal sera). Positive ELISA results were also confirmed by Western blotting analysis. Eight representative ESCC sera which were positive in ELISA had strong reactivity with MDM2 recombinant protein in Western blotting analysis. The same sera also had reactivity with p53 recombinant protein in Western blotting except the serum in lane 8 (Fig. 2). It Table 1 Frequency of autoantibody against MDM2 and p53 in human sera by ELISA. Type of sera ESCC NHS
Number
Autoantibody to MDM2 (%)
Autoantibody to p53 (%)
MDM2 or p53 (%)
157 85
22 (14.0)** 1 (1.2)
36 (22.9)** 0 (0.0)
55 (35)** 1 (1.2)
ESCC, esophageal squamous cell carcinoma; NHS, normal human sera; MDM2, mouse double minute 2. **P < 0.01 (ESCC versus NHS).
Figure 1 Titer of autoantibodies against MDM2 and p53 in esophageal squamous cell carcinoma (ESCC) patients and normal human sera (NHS). The range of antibody titers to MDM2 and p53 is expressed as optical density (OD) obtained from ELISA. The Y-axis represents the OD value. The X-axis represents serum samples. Titer of anti-MDM2 and p53 in ESCC was much higher than that in NHS (P < 0.01).
seemed that there were higher positive rate with MDM2 or p53 antibody reactivity in Western blotting compared with that in ELISA assay. For the possibilities that related to recognition of different epitopes on the same antigen in the ELISA assay and Western blotting, or the different sensitivity between two assays, the results of the two immunological assays varied. Immunofluorescence staining pattern of MDM2 and p53 in Hep-2 cells
To further confirm the reactivity of autoantibodies against MDM2 and p53 in ESCC sera and the intracellular localization of MDM2 and p53, Hep-2 cell slides were used in IIFA to detect ESCC sera with anti-MDM2 and anti-p53 positive in ELISA. As shown in Fig. 3, an anti-MDM2 positive ESCC serum had both cytoplasmic and nuclear staining pattern, while the polyclonal anti-MDM2 antibody with more intense staining in perinuclear regions. The normal human serum had very weak stain. The same serum with anti-p53 had little cytoplasmic and more nuclear staining pattern, while the monoclonal anti-p53 antibody with only intense staining in nuclear regions (Fig. 4). The fluorescent staining was significantly reduced when the same ESCC serum was pre-absorbed with recombinant anti-MDM2 or anti-p53 protein.
Discussion The possible mechanisms underlying the formation of autoantibodies and the regulation of their production remain unknown. Some reports have suggested that this immune response is likely to be related to the dysregulation of the function of these proteins or change of their molecular structure or location during malignant transformation [21]. It was demonstrated that the enhanced B cell toll like receptor 7 expression permitted the specific development of antibodies to RNA/protein complexes and drived anti-RNA autoantibody production [22]. One of the most extensively studied TAAs is p53, which is crucial for cellular stress response and tumour suppression pathways. Sera from cancer patients frequently contain antibodies
Figure 2 Western blotting analysis with representative sera in ELISA. Lane (1) The polyclonal anti-MDM2 antibody and monoclonal anti-p53 antibody were used as positive controls. Lane (2–9) eight representative ESCC sera which were positive in ELISA also had strong reactivity with MDM2 recombinant protein in western blotting analysis. The same sera also have reactivity with p53 recombinant protein except the serum in lane (8).
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A
B
C
Figure 3 Immunofluorescence staining pattern of anti-MDM2 antibody positive in esophageal squamous cell carcinoma (ESCC) serum. (A) A normal human serum (NHS) was used as a negative control. (B) The polyclonal anti-MDM2 antibody that demonstrated both cytoplasmic and perinuclear immunofluorescence staining pattern. (C) A anti-MDM2 antibody positive ESCC serum immunofluorescence staining pattern. (D) The same ESCC serum which used in panel C was pre-absorbed with recombinant MDM2 protein. The fluorescent signal was remarkably decreased.
D
specific for p53 [4, 23]. A study showed that lung tumours containing several types of p53 gene mutations, including missense, stop codon, splice/stop, splice, or frameshift mutations, only the p53 missense mutations, with overexpression of a protein that altered function and increased protein stability, correlated with p53 autoantibody production [24]. It was also demonstrated that there was a statistically significant relationship between p53 autoantibody and the subsequent development of malignancy, and therefore p53 autoantibody can have high predictive value for cancer [13]. Some studies further demonstrated that serum anti-p53 antibodies could be used as early markers for malignant tumours or markers for poor prognosis [25, 26]. p53 plays an important role in cellular physiological and pathological process. It can also form complexes with the MDM2 protein which results in stabilization of p53 [27, 28]. Some patients with antibodies against MDM2 also developed antibodies against p53 [29]. In the sera of Ó 2014 John Wiley & Sons Ltd
ESCC patients autoantibodies against p53 has been detected [30]. Because of the important roles of MDM2 and p53 in cancer formation, we try to validate whether anti-MDM2 and anti-p53 antibodies can be used as biomarkers for early ESCC diagnosis in this study. To test the prevalence of anti-MDM2 and anti-p53 autoantibodies in ESCC patients, recombinant proteins MDM2 and p53 were used as coating antigens in ELISA to detect autoantibodies against the antigens in 157 sera from patients with ESCC and 85 sera from normal individuals. Antibody frequency to individual MDM2 and p53 in ESCC was 14% and 22.9%, respectively. It cannot meet the needs of clinical early diagnosis to ESCC. Both anti-MDM2 and anti-p53 were simultaneously used as diagnostic markers, 35% ESCC cancer patients could be correctly identified, which showed that the clinical diagnostic value of parallel assay of TAAs was high. These data showed the utility of the
366 Autoantibodies to MDM2 and p53 in Esophageal Squamous Cell Carcinoma Y. Chai et al. .................................................................................................................................................................. A
B
C
multiple antigen array in increasing the clinical diagnostic quality and value in cancer. p53 is one of the most frequently mutated genes in cancer. There are multiple ways by which p53 can be regulated. MDM2 is the major regulator of p53 that targets p53 for proteasomal degradation [31, 32]. MDM2 interacts with the transactivation domain of p53 through its N-terminus. MDM2 not only degrades p53 but also regulates its localization. Overexpression of MDM2 is found in a wide variety of human tumours, for example, oral squamous cell carcinomas, breast carcinoma, sarcomas, and leukaemia, due to one of three different mechanisms: gene amplification, increased transcription or enhanced translation [33, 34]. In ovarian carcinomas, 48% of cases showed MDM2 gene amplification [35] However, in ESCC tissues the overexpression of MDM2 was not significantly associated with the abnormal expression of p53 [36]. In this study, we found that some ESCC sera which had strong reactivity with MDM2 recombinant protein also had reactivity with p53 recombinant protein in Western blotting analysis, which indicated that recombinant proteins of MDM2 and p53 as antigens had been shown to be capable of inducing autoantibodies in patients with ESCC. These results were also confirmed by IIFA and it was found that there were strong cytoplasmic and perinuclear expressions of MDM2 and p53 in sera from patients with
Figure 4 Immunofluorescence staining pattern of anti-p53 antibody positive in ESCC serum. (A) Monoclonal anti-p53 antibody that demonstrated a nuclear immunofluorescence staining pattern was used as positive control. (B) A anti-p53 antibody positive ESCC serum mainly nuclear immunofluorescence staining pattern. (C) The same ESCC serum which used in panel b was pre-absorbed with recombinant p53 protein. The fluorescent signal of nuclear immunofluorescence was remarkably decreased.
ESCC. These results indicated that autoantibodies against MDM2 and p53 in ESCC sera might be potential biomarkers for early-stage ESCC screening and diagnosis. Unlike traditional tumour biomarkers, anti-TAA autoantibodies are found in patients’ sera with different cancers, and may represent early indicators of tumour development [37]. The molecular characterization and identification of TAAs in cancers will also contribute to understanding of their functions in malignant transformation, thereby providing attractive candidates for early diagnosis and targeted therapies. Though MDM2 and p53 were overexpressed in many types of cancer tissues and it was demonstrated that MDM2 protein caused the high proliferative activity of ameloblastoma [38], measurement of serum anti-MDM2 antibody was not so much conducted like serum anti-p53 antibody. It was also found that serum anti-p53 antibody measurement does not contribute to the diagnosis of pancreatobiliary cancers [39]. Therefore, identification of novel serum biomarkers for the diagnosis of cancer, especially for detection and screening in earlystage cancer, is an important means to improve clinical outcome [40]. In conclusion, a total of 157 sera from patients with ESCC have been tested with different immunological assay. The data in our study suggests that MDM2 and p53 can induce autoantibody responses in sera from patients with
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ESCC, indicating that anti-MDM2 and anti-p53 autoantibodies might be potential biomarkers for clinical serologic screening in the diagnosis of ESCC. Further studies to elucidate the mechanism of anti-MDM2 and anti-p53 autoantibodies production will be necessary for a better understanding of the immune status in ESCC.
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Acknowledgment
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The authors thank Dr. Eng M. Tan (The Scripps Research Institute, USA) for his support. This work was supported by grants from the National Natural Science Foundation of China (81372371), and also by a grant (SC1CA166016) from the National Institutes of Health of USA (NIH).
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Conflict of interest
21
The authors declare that there is no conflict of interests regarding the publication of this paper.
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