Differential expression of anterior gradient gene AGR2 in prostate cancer

Abstract

Background: The protein AGR2 is a putative member of the protein disulfide isomerase family and was first identified as a homolog of the Xenopus laevis gene XAG-2. AGR2 has been implicated in a number of human cancers. In particular, AGR2 has previously been found to be one of several genes that encode secreted proteins showing increased expression in prostate cancer cells compared to normal prostatic epithelium.

Methods: Gene expression levels of AGR2 were examined in prostate cancer cells by microarray analysis. We further examined the relationship of AGR2 protein expression to histopathology and prostate cancer outcome on a population basis using tissue microarray technology.

Results: At the RNA and protein level, there was an increase in AGR2 expression in adenocarcinoma of the prostate compared to morphologically normal prostatic glandular epithelium. Using a tissue microarray, this enhanced AGR2 expression was seen as early as premalignant PIN lesions. Interestingly, within adenocarcinoma samples, there was a slight trend toward lower levels of AGR2 with increasing Gleason score. Consistent with this, relatively lower levels of AGR2 were highly predictive of disease recurrence in patients who had originally presented with high-stage primary prostate cancer (P = 0.009).

Conclusions: We have shown for the first time that despite an increase in AGR2 expression in prostate cancer compared to non-malignant cells, relatively lower levels of AGR2 are highly predictive of disease recurrence following radical prostatectomy.

Figure 1

AGR2 transcript expression in prostate cancer. Two publically available datasets were used to examine AGR2 gene expression in human prostate samples [31,32]. (A) Dataset was from Yu, et al., and was generated using Affymetrix U95B Array [31]. (B) Dataset was from Lapointe, et al., and was generated using a cDNA microarray from the Stanford Microarray Database [32]. For each dataset, normalized data was available from the GEO database. The bars are the relative mean expression value ± standard error of the mean. P < 0.0001 for each multi group comparison (Kruskal-Wallis).

Figure 2

AGR2 immunohistochemistry. AGR2 staining was localized to the cytoplasm and membrane of cancer epithelial cells. (A) Tissue samples incubated with concentration-matched non-immune rabbit IgG showed no staining. Shown are representative sections of (B) morphologically normal tissue, (C) PIN, and (D) a representative adenocarcinoma (Gleason grade 7) showing low staining intensity, (E) a representative adenocarcinoma (Gleason grade 8) showing moderate staining intensity, and (F) a representative adenocarcinoma (Gleason grade 6) showing high staining intensity.

Figure 3

AGR2 expression by spot level histopathology. The barplots show the mean integrated AGR2 expression by TMA spot-level histology in morphologically normal (NL), BPH, PIN, adenocarcinoma (AD), and lymph node metastases (LNMET); bars are standard errors. Levels of AGR2 expression were increased in PIN lesions and adenocarcinoma compared to BPH and morphologically normal adjacent tissue (P < 0.0001). Regional metastases showed increased AGR2 expression compared to BPH and normal tissue (P = 0.027 and P = 0.030, respectively).

Figure 4

AGR2 expression by spot level Gleason grade. The barplots show the mean integrated AGR2 expression by TMA spot-level Gleason grade; bars are standard errors. Average AGR2 expression was increased in grades 3 and 4 compared to grade 2 (P < 0.0001 and P = 0.0007) and grade 5 (P = 0.0028 and P = 0.0085).

Figure 5

AGR2 levels predict prostate cancer recurrence in high stage patients. Solid line is relatively higher AGR2 levels (staining measure ≥0.79); dashed line is relatively lower AGR2 levels (< 0.79). In men with stage III or IV prostate cancer, relatively lower levels of AGR2 predict a significantly greater chance of prostate cancer recurrence compared to those with higher levels (P = 0.009).