It is estimated that in 2010, there will be over 217,000 new cases of prostate cancer and over 32,000 deaths from this disease in the United States alone, making it the most common male cancer and the second leading cause of cancer-related deaths in men [1]. Prostate cancer is generally diagnosed by serum level of prostate-specific antigen (PSA) and digital rectal exam. However, although PSA is abundantly synthesized by the prostate, it is not cancer specific, leading to many unnecessary biopsies. Improved markers for both identifying prostate cancer and predicting its outcome are needed. Our approach to discover such markers involved comparative analysis of the transcriptomes of cancer cells and normal cells. These transcriptomes were determined through sorting of specific cell types from appropriate tissue specimens for analysis by Affymetrix DNA microarrays [2]. Genes that were found overexpressed by 8-fold or more in cancer cells were biomarker candidates, especially those that encode secreted or extracellular proteins. The coding sequences were analyzed with SignalP 2.0 [3] for signal peptides and TMHMM [4] for protein topology and the number of transmembrane helices. AGR2 (anterior gradient 2) was one among several such genes identified as overexpressed in prostate cancer cells. The array signal level for AGR2 in the cancer cells was ~50-fold higher than that in luminal cells, the normal counterpart [5].

AGR2 is the human homolog of the protein XAG-2 in Xenopus laevis and was first identified as differentially expressed in estrogen receptor positive breast cancer cell lines [6]. Biochemically, AGR2 is classified as a member of the protein disulfide isomerase (PDI) family based on amino acid sequence homology [7]. PDI enzymes in the endoplasmic reticulum act as molecular chaperones for protein folding. As such, AGR2 is thought to act as a chaperone to clear misfolded proteins out of cells during periods of physiological stress [8]. Studies have found that AGR2 expression can be increased in response to physiological stress in breast cancer cells and can enhance survival of damaged lung cancer cells [8, 9]. PDI enzymes are found in other subcellular compartments and are thought to participate in or modulate a range of functions, from cell adhesion to DNA binding [10–12].

Several cancers, including breast [13–16], prostate [17–21], fibrolamellar [22], pancreatic [23–25], and colon [26], have been found to express increased levels of AGR2 compared to normal tissue. Furthermore, AGR2 has been shown to increase tumor cell migration in vitro and the incidence of metastatic lesions in vivo [15, 18, 21, 27]. Accordingly, some studies have found AGR2 to be a marker of poor prognosis in human breast and prostate cancers [14, 19, 28, 29]. One the other hand, some studies have found AGR2 to show no association with patient outcome in lung and pancreatic cancer [23, 30] or even improved outcome in breast cancer [13].

To further evaluate the utility of AGR2 as a cancer biomarker, we examined the expression pattern of AGR2 in prostate cancer using both gene expression analysis and a high-density tissue microarray.

AGR2 has been implicated in cancer pathogenesis and has been found to be up-regulated in multiple human cancers, including breast, lung, and prostate [13, 14, 18–20, 30]. Our study has shown that AGR2 is higher in prostate cancer cells compared to non-malignant prostatic epithelial cells at the transcript and protein levels. This is consistent with other recent studies which have found an increase in AGR2 mRNA using microarray analysis of laser-capture microdissected cells and an increase in AGR2 protein levels [17–21, 39, 40]. Using TMA technology, we also verify a greatly enhanced AGR2 protein expression in malignant and early malignant PIN lesions compared to non-malignant epithelium. We observed that AGR2 levels were lower in advanced disease states as Gleason grade 5 spots expressed dramatically less AGR2 than grade 4 and grade 3 spots. Finally, we showed, for the first time, a novel predictive value for AGR2 expression with regard to tumor recurrence in individuals with higher stage disease. In this situation, relatively lower levels of AGR2 expression predicted a higher likelihood of tumor recurrence.

The exact biological role of AGR2 in humans is largely unknown. The AGR2 homolog in X. laevis, XAG2, has 73% similarity and 47% identity at the protein level [6] and plays a role in the differentiation of the mucus-secreting cement gland found on the anterior aspect of the frog embryo [41]. The newt homolog, nAG, has been shown to induce cellular proliferation in denervated limbs [42]. In the adult, AGR2 expression is restricted to a limited number of tissues and cell types in the body suggesting it is not a ubiquitously expressed product [43]. In the intestine, for example, AGR2 is found in the goblet cells and appears necessary for the production of intestinal mucus [44, 45]. In malignant cells from organs such as the breast and prostate, AGR2 may be involved in additional functions. Interestingly, in various models of tumor progression, increased AGR2 promotes cell migration, invasion, and presumably metastatic spread [15, 18, 21, 27, 28, 46, 47]. However, the story may be more complicated as recently Bu, et al. observed that while over-expressed AGR2 in prostate cancer cells increased migration and invasion, it additionally repressed growth and proliferation [21]. The exact mechanistic role for AGR2 in these functions remains to be elucidated.

In our data, the lower expression of AGR2 in higher Gleason tumors appears to correlate with AGR2 being predictive of prostate cancer recurrence in individuals with higher stage cancers. Many studies report that AGR2 expression corresponds to a more differentiated phenotype [13, 30, 46]. Lower Gleason scores are indicative of well-differentiated tumors. If AGR2 indeed has opposing functions of promoting migration and invasion with higher expression yet allowing/promoting cellular proliferation upon decreased expression [21], it is intriguing to consider that our results reflect this balance of functional activity. We therefore hypothesize that in later stage prostate cancer, the balance towards increased proliferation (e.g., lower AGR2 expression) outweighs the need for enhanced migration and invasion (e.g., higher AGR2 expression). We are currently testing this hypothesis both in vitro and in vivo. Of interest Zhang et al., also used TMA technology to examine the associate of AGR2 expression levels with prostate cancer outcome [19]. Similar to us, they observed an increase in AGR2 expression in prostate cancer compared to normal or benign tissue. However, in apparent contrast to our results, they observed that increased AGR2 was associated with a poorer outcome [19]. While the explanation for this apparent discrepancy is unclear, it should be noted that Zhang et al. considered overall survival while our outcome measure was disease-specific recurrence. We did not have sufficient numbers of patients in our cohort who died from the disease in order to conduct meaningful statistical analysis of survival. Whether or not other subtle differences in treatment, clinical history, and/or demographics existed in the patient cohort at UCLA versus the Royal Liverpool University Hospital is difficult to assess. Nevertheless, whether the prognostic significance of AGR2 varies based on the outcome measurement (i.e., disease recurrence versus survival) warrants further investigation.

Our group, as well as others, is also examining AGR2 as a potential prostate cancer biomarker. In this regard, AGR2 has the following interesting attributes. First, it appears to be expressed at relatively high levels in individuals with prostate cancer; expression is lower from normal or non-malignant prostatic epithelium. Second, since AGR2 is secreted, there is the likelihood that the protein can be detected in blood or urine. Indeed Bu et al., have detected increased AGR2 transcript in urine sediments from prostate cancer patients [21]. That AGR2 is secreted is supported by our results as well as by mass spectrometry proteomic analysis of 2-D gel electrophoresis spots reported in the literature [48]. We are currently exploring whether AGR2 detected in body fluids is an accurate gauge for prostate cancer initiation, progression, and/or outcome. In particular, we are developing an assay to detect subnanogram per ml levels of AGR2. New monoclonal AGR2 antibodies need to be produced, however, since we found that the commercially available one did not recognize native AGR2 in solution.

In summary, we found that AGR2 is a secreted protein expressed at relatively high levels by prostate cancer cells and cells in PIN lesions. We found that a relatively higher expression of AGR2 in patients with high stage prostate cancer stratified these individuals as less likely to have a tumor recurrence.