We recently identified leucine-rich alpha-2-glycoprotein-1 (LRG1) as one of several proteins overexpressed in the serum of patients with ovarian cancer [8, 9]. In this study, we sought to validate this observation and quantitate the levels of LRG1 in a larger cohort of patients' sera. We have also shown that ovarian cancer cells may directly contribute to the elevated levels of LRG1 observed in patients' sera.
The increased serum LRG1 levels in ovarian cancer patients that we had observed by Western blot in pooled samples , were also evident by ELISA in individual samples. When the initial 114 serum samples were tested by ELISA, serum LRG1 was found to be approximately 2-fold greater in serous ovarian cancer patients' sera compared to sera from healthy control women; however, the variance among the ovarian cancer patient samples resulted in unfavorable estimates of sensitivity and specificity. This led us to explore the levels of serum LRG1 among women with different types of benign and malignant ovarian masses. Using a separate set of 193 patient serum samples obtained immediately prior to surgery for a suspicious adnexal mass, LRG1 values were significantly higher (1.7-fold) in patients with serous and clear cell ovarian cancer compared to those with benign gynecological diseases. Although our gene microarray data showed that LRG1 mRNA expression levels were greatest in low malignant potential tumors, the level of serum LRG1 protein in the LMP tumors was significantly lower than for both serous and clear cell ovarian cancer.
Although a biomarker for the early detection of ovarian cancer would have a greater impact, the ability to distinguish malignant from benign disease prior to surgery could be useful in determining which patients would benefit from treatment by a gynecological oncologist. Recently, a panel of biomarkers was approved by the FDA to aid in the diagnosis of ovarian tumors prior to surgery (OVA1; ). This panel includes CA125 as well as beta-2 microglobulin, apolipoprotein A1, transthyretin and transferrin, but not LRG1.
Though the mean concentration of serum LRG1 in serous ovarian cancer patients differed between samples in the two data sets, differences in serum preparation and storage may have affected the quantity of LRG1 detected. For example, Govorukhina et al.,  recently reported that LRG1 levels were decreased in serum with clotting time of longer than 1 hr. We maintained a strict protocol for sample collection and storage for the samples taken from patients at the University of Minnesota, in order to minimize these types of variations (see Methods), and this likely explains the higher LRG1 values in the second dataset compared with the first set of samples obtained from the GOG.
Initially, LRG1 was classified as an "acute-phase protein" involved in the body's response to bacterial and viral infection , but has since been identified as elevated in a variety of disease states, both malignant and benign, including toxic-shock syndrome , and during inflammatory responses of cystic fibrosis . LRG1 is also increased in serum of patients with hepatocellular carcinoma following therapeutic ablation treatment . Differential expression techniques employing affinity depletion of high abundance proteins and 2 D electrophoresis have found serum LRG1 to be upregulated in lung and pancreatic cancer [35–37]. Proteomic research using 2 D SDS-PAGE to analyze body fluids found LRG1 to be upregulated in cerebrospinal fluid and serum of patients with hydrocephalus and silicosis [19, 38].
We conducted a series of experiments examining ovarian cancer tumor cells as a possible source of serum LRG1. Others have identified LRG1 peptides by mass-spectrometry in the secreted or cell surface fractions of CAOV3 and OVCAR3 serous ovarian cancer cell lines, but not in the clear cell ovarian cancer cell line ES-2 . LRG1 peptides have also been identified in ascites fluid and cells from ovarian cancer patients . Recently, elevated levels of LRG1 have been identified in chemoresistant ovarian tumor tissue , and in immunodepleted serum, using ICAT quantitative proteomics . Additionally, LRG1 peptides have been identified in the conditioned media of prostate cancer [43, 44], and breast cancer cell lines  and in the peritoneal fluid of women with uterine leiomyomas . The production and secretion of LRG1 by tumor cells suggests there may be a more direct relationship between tumor burden and serum levels of LRG1 than for other acute phase proteins secreted only by the liver. For example, although haptoglobin levels are increased in the sera of ovarian cancer patients, no haptoglobin RNA or protein were detected by Ye et al.  in seven ovarian cancer cell lines.
In a limited number of cases, we have analyzed sera from patients prior to surgery and following treatment for ovarian cancer. We have found that serum LRG1 levels appear to be more directly related to tumor burden compared to CA125. For example, in three of six patients with sub-optimal debulking surgery, CA125 levels dropped substantially, while LRG1 levels remained elevated. In six cases, serum LRG1 dropped dramatically post chemotherapy. In five cases, LRG1 levels appeared to rise prior to CA125 levels and the onset of recurrent disease. However, given the very low numbers of patients that we have analyzed to date, the use of LRG1 as a marker for disease recurrence, while tantalizing, is purely speculative.
By immunocytochemistry, LRG1 was localized to the cytoplasm of all of the ovarian cell lines tested, both cancer and normal, and was observed on the plasma membrane of most. The serous papillary ovarian cancer cell line, NIH:OVCAR5, had the most intense plasma membrane staining for LRG1. In addition, this cell line expressed high levels of the ~51 kD LRG1 protein band. The LRG1 sequence contains a predicted transmembrane domain  which overlaps the signal sequence; this may allow for the expression of LRG1 at the cell surface. Alternatively, the localization of LRG1 that we observed on the surface of the NIH:OVCAR5 cells may be indicative of cells in the process of secreting LRG1.
Examination of ovarian tumor extracts and cell lines by Western blot revealed increased expression of LRG1 protein in malignant serous tumors and ovarian cancer cell lines compared to their respective controls, as well as the presence of several isoforms of LRG1, though notably the ~47 kD LRG1 band was most intense in each of the malignant ovarian tumor protein extracts. The presence of numerous isoforms for LRG1 has previously been shown by 2 D SDS-PAGE [8, 11, 32, 34, 36–38], suggesting the presence of multiple glycosylated isoforms of LRG1. Indeed, we showed that glycosidase treatment of LRG1, both purified and in extracts of ovarian cancer and NOSE cell lines, reduced the apparent molecular weight of LRG1 indicating the presence of carbohydrate modifications of the protein backbone.
The N-glycosylation of LRG1 produced by the ovarian cancer cells is consistent with its secretion. The ~51 kD band was found at very low levels in the ovarian cancer tumor extracts and was present in the protein extracts of only a few of the ovarian cancer cell lines. It is possible that this ~51 kD glycoform of LRG1 is secreted by the serous ovarian cancer cells, and may contribute to the elevated levels of LRG1 quantitated by ELISA in the sera of these patients. This hypothesis is supported by our Western blot findings that an ~51 kD band was found in the conditioned media of the NIH:OVCAR5 cells but not the NOSE cells, again suggesting that the ~51 kD glycoform of LRG1 may be preferentially secreted, or aberrantly glycosylated in ovarian cancer.
Glycosylation of serum proteins in cancer states is well documented, and serum glycoproteins are being investigated for use as biomarkers in prostate, breast, lung, ovarian and other gynecologic cancers [49–52]. Glycosylation of surface proteins on ovarian carcinoma cells has been reported to mediate adhesion, migration, and invasion through the ECM . Given that murine LRG1 has been shown to bind to several extracellular matrix proteins, and also TGFβ , a possible role for LRG1 in ovarian cancer progression is intriguing.
Alternatively, LRG1 may be playing a role in apoptosis. We have found that MCF-7 breast cancer cells transfected with LRG1 are more resistant to apoptosis induction than non-transfected cells due to cytoplasmic LRG1 binding cytochrome c and inhibition of Apaf-1 activation (Jemmerson and colleagues, manuscript in preparation). In addition, transformed granulocytic cells transfected with LRG1 were reported by Ai et al.  to be more viable than non-transfected cells when transferred between different media. Likewise, LRG1 may be a survival factor for ovarian cancer cells, possibly rendering them more resistant to chemotherapy. It is interesting to note that the cisplatin-resistant A2780-CP cells express higher levels of LRG1 protein than their more sensitive counterparts A2780-S (Figure 3B); however, no difference in LRG1 protein expression was found for the cisplatin resistant cell line C13, compared to the corresponding cisplatin sensitive cell line OV2008.