Cancer cells are known to acquire the hallmarks of malignancy through the accumulation of advantageous gene activation and inactivation events over long periods of time (Hanahan and Weinberg, 2000
). To identify a novel ovarian tumor marker for early detection, pooled scrapings of human normal ovarian surface epithelium rather than whole ovaries were used to compare gene expression with ovarian tumors. Previously, AGR2 was reported to be expressed in cancers of the breast (Fritzsche et al., 2006
), pancreas (Ramachandran et al., 2008
; Riener et al., 2009
), and prostate (Zhang et al., 2005
). Overexpression or suppression of AGR2 was shown to affect cell proliferation, cell invasion, metastasis, and tumor growth of pancreatic cancer (Ramachandran et al., 2008
). Recent reports support a role for AGR2 in cellular transformation and growth of esophageal adenocarcinoma (Wang et al., 2008
Our systematic approach combining cDNA microarray analysis, RT-PCR, immunohistochemistry, Western blot analyses, and bioinformatics facilitated the identification of AGR2 as a novel candidate of an ovarian tumor marker for the early detection of mucinous ovarian tumors. More importantly, in terms of clinical usefulness, we were able to detect AGR2 in patient serum by Western blot and ELISA assays. Detection in patient serum by an ELISA assay developed in this study provides an efficient screening of mucinous ovarian tumors among other ovarian tumors. Cumulative molecular genetic analyses of ovarian cancers have revealed genetic alterations of several genes in ovarian tumors, such as c-erb-B2, c-myc, and p53 (Aunoble et al., 2000
). However, none of these mutations are diagnostic of malignancy. Previous reports by other investigators have identified multiple molecular targets that differ in normal and malignant cells in epithelial ovarian cancer, including cell cycle regulators, growth factor receptors, signal transduction pathways, genes that regulate drug resistance, and angiogenesis (reviewed in See et al., 2003
; Sharma and Odunsi, 2005
). In addition, AGR2 was shown to be a useful marker for detection of circulating tumor cells (CTCs) in peripheral blood of advanced cancer patients (Smirnov et al., 2005
). The AGR2 protein has been previously associated with breast carcinomas by suppression subtractive cDNA library screening between a malignant human breast cancer cell line (MCF-7) and a benign breast tumor-derived cell line (Huma 123; Liu et al., 2005
). Screening of breast tumor specimens using RT-PCR and immunocytochemistry with anti-AGR2 antibodies has demonstrated that the presence of AGR2 mRNA and protein is statistically and significantly associated with ERα-positive carcinomas and malignancy (Fritzsche et al., 2006
). In addition, expression of AGR2 has been reported to be strongly associated with markers of tumor differentiation (estrogen receptor positivity and lower tumor grade). Although CA-125 is the best clinical marker for ovarian cancer in the postmenopausal age group, CA-125 has proven to be a poor diagnostic tumor biomarker for early stage ovarian cancer (Liede et al., 2002
). Importantly, the CA-125 level was not elevated in the mucinous subtype. Previous efforts to characterize serum biomarkers for the detection of early-stage ovarian cancer identified transthyretin, beta-hemoglobin, apolipoprotein A1, and transferrin using micro-LC-MS/MS (Kozak et al., 2005
). Unfortunately, the combination of CA-125 with biomarkers, such as transthyretin, beta-hemoglobin, apolipoprotein A1, and transferrin, did not further improve the detection of mucinous tumors (Kozak et al., 2005
). There has been an increase in the search for ovarian cancer markers using SELDI-TOF-MS with the use of multiple markers (Zhang et al., 2004
), but there is still no significant improvement for detection of mucinous ovarian cancer. Considering the preferential expression of borderline mucinous cancer, AGR2 would be beneficial to improve the early detection of mucinous ovarian tumors.
To gain insight into the role of AGR2 in ovarian tumorigenesis, AGR2-overexpressing stable human ovarian cancer cells were generated and our results showed that AGR2 enhanced cell growth and migration of ovarian cancer cells. Consistent with the involvement of AGR2 in cell growth and migration, our cDNA microarray analysis of AGR2-stable cells revealed that AGR2 overexpression up-regulated the expression of genes involved in cell proliferation, invasion, and angiogenesis, which is important for the tumor progression and metastasis characteristic of ovarian cancer. By contrast, the genes involved in the negative regulation of cell proliferation, cell adhesion, and cell death were down-regulated. Moreover, AGR2 protein can be detected in the serum of mucinous ovarian tumor patients by Western blot and ELISA methods. These results suggest that serum AGR2 can be a biomarker for diagnosis and prediction of mucinous ovarian cancers. This is the first report that AGR2 is a candidate molecular marker for mucinous ovarian tumors. However, validation of AGR2 as a potential serum marker is needed with a large-scale analysis of ovarian cancer blood samples and can be compared with other validated serum markers. We continue to expand our effort to collect serum from mucinous ovarian cancer patients and normal volunteers. Because AGR2 has previously been reported to be associated with tumor metastasis, it would be interesting to determine the role of AGR2 in ovarian tumor progression and metastasis. Analysis of AGR2 protein suggests potential protein modification that might be important for AGR2 function (Park and Lee, unpublished data). Further functional analysis of AGR2 protein modification during tumor progression would provide information for its functional significance in addition to its role in diagnosis. Recently AGR2 was classified as a member of the protein disulfide isomerase (PDI) family and shown to play a special role in the production of mucus (Park et al., 2009
). AGR2 is also expressed in mucus-producing cells, and AGR2-deficient mice were unable to produce intestinal mucin and became highly susceptible to experimentally-induced colitis.
The impact of results in this study on cancer biomarker discovery efforts is significant because ovarian cancer remains a highly fatal cancer due to the lack of reliable screening tests and high recurrence of ovarian cancer. The results from this study suggest that AGR2 can be a potential biomarker for diagnosis of mucinous ovarian cancer and the ELISA assay would facilitate the early detection of mucinous ovarian cancer using a non-invasive method using patient serum.