Int J Clin Exp Pathol 2014;7(7):3918-3927 /ISSN:1936-2625/IJCEP0000852

Original Article Overexpression of MAGE-D4 in colorectal cancer is a potentially prognostic biomarker and immunotherapy target Qing-Mei Zhang1*, Shu-Jia He1*, Ning Shen2*, Bin Luo1, Rong Fan1, Jun Fu1, Guo-Rong Luo1, Su-Fang Zhou1, Shao-Wen Xiao3, Xiao-Xun Xie1 Department of Histology and Embryology, School of Pre-clinical Medicine, 3Department of Surgery, First Affiliated Hospital, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China; 2Department of Oral and Maxillofacial Surgery, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, Guangxi Zhuang Autonomous Region, China. *Equal contributors. 1

Received May 20, 2014; Accepted June 27, 2014; Epub June 15, 2014; Published July 1, 2014 Abstract: Melanoma-associated antigen D4 (MAGE-D4) is a novel member of MAGE family. This study aimed to examine the expression and immunogenicity of MAGE-D4 in colorectal cancer (CRC) to determine its potential as a prognosis and immunotherapeutic target. The expression of MAGE-D4 mRNA and protein was determined by RT-PCR and immunohistochemistry (IHC) in CRCs with paired adjacent non-tumor tissues, colorectal adenomas and normal colorectal tissues, respectively. Sera from 64 CRC patients were tested for MAGE-D4 antibody by ELISA. MAGED4 mRNA was more frequently expressed in CRCs (76.7%, 46/60) than in adjacent non-tumor tissues (15.0%, 9/60). MAGE-D4 protein was detected in all the CRC tissues tested, 70.0% of which showed high expression. There was no MAGE-D4 protein detected in any paired adjacent non-tumor tissue. No MAGE-D4 expression was found in colorectal adenomas and normal colorectal tissues by either RT-PCR or immunohistochemistry. Patients with high MAGE-D4 protein expression had significantly shorter overall survival than those with low MAGE-D4 protein expression (median, 68.6 vs 122.2 months; P=0.030). Furthermore, multivariate analysis exhibited high MAGE-D4 protein expression had a trend toward an independent prognostic factor (hazard ratio: 6.124; P=0.050). Humoral immunity to MAGE-D4 was detected in 12 of 64 (18.8%) CRC patients’ sera but not in 77 healthy donors. There was no correlation between MAGE-D4 expression, serum antibody and clinicopathological parameters. These findings suggest MAGE-D4 may serve as a potentially prognostic biomarker and an attractive target of immunotherapy in CRC. Keywords: Melanoma-associated antigen, MAGE-D4, colorectal cancer, serum immunoreactivity

Introduction Colorectal cancer (CRC) is the third most common malignancy and fourth leading cause of cancer mortality worldwide, with more than a million individuals diagnosed and about half million deaths annually [1, 2]. Although there are many established therapeutic strategies including surgery, chemotherapy and radiotherapy, its prognosis remains unsatisfactory due to late diagnosis [3]. Thus, novel therapeutic strategies are urgently needed for this malignancy. Immunotherapy is an attractive approach among novel therapeutic strategies [4, 5]. This approach requires the identification of tumor specific antigens. Currently, a number of such antigens are encoded by the genes of

melanoma-associated antigen (MAGE) family [6, 7]. MAGE is a large gene family including more than 60 members, in which some genes encode tumor-specific antigens with characteristics of broad expression in various tumors but restricted in normal tissues, and recognized by cytotoxic T lymphocytes (CTLs) [8, 9]. Some of MAGE antigens and their epitope peptides constitute important targets for antitumor immunotherapy with a number of clinical studies already completed or underway [10-12]. Unfortunately, It has been found that CRC expressed some of MAGE antigen with low frequency, especially those (Mage-A1 and -A3) which had been applied on clinical trial [13-18]. Therefore, iden-

MAGE-D4 in colorectal cancer tification of other MAGE antigens with high expression in CRC is essential. MAGE-D4, originally named MAGE-E1, is a novel member of MAGE family. It has been reported restricted expression in normal tissues except brain and ovary and overexpression in some human malignancies, including lung, liver, oral, kidney, breast, esophagus cancer and glioma [19-25]. A MHC class I ligand from MAGE-D4 presented by HLA-A on tumor tissue was also reported by Kramer et al [20]. Several previous studies have shown that MAGE-D4 contributes to proliferation, migration, and invasion of tumor cells in breast cancer and oral squamous cell carcinoma [22, 23]. Recent studies have shown that MAGE-D4 is a marker of poor prognosis in hepatocellular, esophagus and breast carcinoma [23-25]. To some extent, these preliminary findings have been suggested that MAGE-D4 may play an important role in the progression of tumors and may be a potentially promising target for tumor prognosis and treatment. Current knowledge about MAGE-D4 gene expression in CRC was only based on mRNA level from one previous report [26]. However, MAGE-D4 protein expression has not been elucidated including its immunogenicity. In the present study, we examined the expression of MAGE-D4 at mRNA and protein levels in CRC tissues, as well as the serum antibody against MAGE-D4 in a subset of CRC patients. Furthermore, whether MAGE-D4 protein can be a prognostic factor was analyzed, and the correlation among MAGE-D4 expression, serum antibody and clinicopathological parameters in CRC patients were also investigated. Materials and methods Tissue and serum samples All tissue and serum samples were collected from the First Affiliated Hospital of Guangxi Medical University with the informed consent of patients and approved by Hospital Ethic Review Committee. A total of 154 tissue samples, including 60 primary CRCs with adjacent nontumor tissues (42 men and 18 women; mean age, 56.37±14.74 years; age ranging, 30-86 years), 24 colorectal adenomas and 10 normal colorectal tissues, were analyzed by RT-PCR. 82 of paraffin-embedded tissue sections for immunohistochemical analysis was construct3919

ed from 30 primary CRCs with paired adjacent non-tumor tissues (17 men and 13 women; mean age, 56.45±11.43 years; age ranging, 35-76 years), 12 colorectal adenomas and 10 normal colorectal tissues. CRC patients were followed up for 1-130 months (mean, 79.6±52.6 months). Overall survival was defined as the time from diagnosis to the date of death or last follow-up. All CRC and paired non-tumor tissues were surgically removed without undergoing preoperative treatment including chemotherapy and radiation. The colorectal adenomas were available from the patients by endoscopic polypectomy and normal colorectal tissues were obtained at autopsy. Sera were collected from 64 patients with CRC (47 men and 17 women; mean age, 56.03±14.96 years; age ranging, 26-93 years) at diagnosis prior of therapy and sera of 77 healthy donors from routine physical examination of students of Guangxi Medical University were used as controls. All tumor samples were classified according to the TNM classification of the Union for International Cancer Control [27]. Tumor samples were coded and assessed in a blinded manner. RT-PCR analysis Total RNA was prepared by Trizol reagent (Invitrogen, CA) and 2.5 μg RNA was reverse transcribed into cDNA with RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, USA) according to the manufacturer’s instructions. cDNA was then tested for integrity by amplification of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. RT-PCR was carried out with MAGE-D4 specific primers as previously reported [19]. The cycling parameters were as following: initial denaturation at 94°C for 5 min followed by 30 sec at 94°C, 30 sec at (64°C for MAGE-D4, 55°C for GAPDH), and 30 sec at 72°C for 35 cycles, and a final extension for 10 min at 72°C. Target bands were visualized on a 1.5% agarose gel with ethidium bromide staining. The expression of MAGE-D4 was counted as positive, only if the RT-PCR reaction repeated at least twice with same result. Immunohistochemistry (IHC) Immunohistochemistry (IHC) was performed with minor modification as previous report by Luo et al [28]. In brief, formalin-fixed, paraffinembedded tissue sections were deparaffinized and rehydrated under the regular condition. Subsequently, the sections were heated in ethInt J Clin Exp Pathol 2014;7(7):3918-3927

MAGE-D4 in colorectal cancer ELISA was performed as previous report by Zhou et al [31]. In brief, MAGE-D4 protein (1 μg/ml) was coated on the 96-well plates (Corning, USA) at 4°C overnight. Maltose binding protein (MBP) protein was used as a blank control. The plates were blocked with 5% nonfat milk 37°C for 1 h, then 1:800 diluted Figure 1. RT-PCR analysis of MAGE-D4 mRNA expression in CRC tissues (T) with serum were added and paired adjacent non-tumor tissues (Nt), colorectal adenomas (A) and normal colorectal tissues (N). GAPDH was used as internal control for the parallel PCR incubated at 37°C for 1 h. analysis of the same sample. P, positive control (glioma); Ne, negative control Streptavidin-biotinylated (no cDNA template). horseradish peroxidase complex (KPL, USA) was ylene diamine tetraacetic acid (EDTA, pH8.0) used as secondary antibody. Detection was accomplished using tetramethylbenzidine subfor antigen retrieval. After the inactivation of strate, followed by adding sulfuric acid to stop endogenous peroxidase, the sections were the reaction. The absorbance at 450 nm with treated with normal goat serum for blocking 630nm as reference filter was determined with and then immunostained with anti-MAGE-D4 a microplate reader (Bio-Rad, USA). All serum polyclonal antibody (1:500 dilution, Santa Cruz samples were performed from triplicates and Biotechnology, USA) overnight at 4°C. Negative indicated as mean. An optical density (OD) controls using rabbit serum collected before value that exceeds three standard deviations immunization were also incubated in parallel. (SDs) above the mean OD value of sera from Then horseradish peroxidase-conjugated goat healthy donors was defined as positive. anti-rabbit IgG (ZSGB-BIO, China) was added as Specificity of each positive serum sample was the secondary antibody. Immunoreactivity was examined by testing reactivity after pre-incuvisualized with 3, 3’-diaminobenzidine (DAB) bating with recombinant MAGE-D4 protein. (Maixin Biotechnology, China) followed by hematoxylin counterstain. Statistical analysis Positive immunoreactivity was assessed by two Statistical analyses were conducted by SPSS independent pathologists who did not know software (version 16). Statistical significance patients’ clinical information and recorded was defined as P

Overexpression of MAGE-D4 in colorectal cancer is a potentially prognostic biomarker and immunotherapy target.

Melanoma-associated antigen D4 (MAGE-D4) is a novel member of MAGE family. This study aimed to examine the expression and immunogenicity of MAGE-D4 in...
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