Low expression and significance of AOC1 in prostate adenocarcinoma

Introduction

Amine oxidase copper-containing 1 [AOC1, formerly known as amiloride-binding protein 1 (ABP1)], is a secreted glycoprotein that can catalyze the degradation of putrescine and histamine. Polyamines and their diamine precursor, putrescine, are omnipresent in all organisms and play key roles in cellular growth and proliferation (1). AOC1 is a pivotal degrading enzyme in mammals that catalyzes the metabolism of histamine and putrescine, thereby playing a crucial role in regulating the homeostasis of biogenic amines (2). However, there are few studies on AOC1 in prostate cancer. This study was designed to investigate the expression profiles of AOC1 in prostate cancer and normal prostate tissues with an attempt to provide novel evidence for the diagnosis and treatment of prostate cancer. We present this article in accordance with the REMARK reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-258/rc).

Methods

Clinical data

The clinicopathological data of 101 patients diagnosed with prostate adenocarcinoma at the Pathological Diagnosis Center of the Third People’s Hospital of Bengbu between May 2017 and June 2024 were retrospectively collected. Additionally, adjacent prostate tissues from 22 patients were harvested and used as the normal control (NC) group. These patients were aged 54 to 95 years, with a median age of 75 years. Follow-up was conducted via telephone, with a final follow-up date set for January 2024; recurrence was primarily clinical recurrence, combined with serum prostate specific antigen (PSA) levels (biochemical recurrence).

From January 2024 to March 2024, urine and blood samples were collected for the detection of the AOC1 protein level,18 patients with prostate cancer (aged 59–83 years, median age 70.5 years), and 6 patients at 3 months post-prostatectomy for prostate cancer (aged 60–79 years, median age 71 years); due to certain reasons, blood samples are missing from 4 patients. In order to better assess the expression significance of AOC1 in prostate cancer, and to comprehensively understand the expression profile of AOC1 in the urinary system and male reproductive system tissues, we also collected 27 samples of these systems. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the ethics committee of the Third People’s Hospital of Bengbu (ethical No. 2023-01). Individual consent for this retrospective analysis was waived.

Immunohistochemistry

The anti-ABP1 (AOC1) antibody (EPR24299-52; Abcam, UK) was used at a dilution of 1:100 for immunohistochemical analysis. The tissue sections were treated with an ethylenediaminetetraacetic acid (EDTA)-based repair solution for heat- and pressure-induced antigen retrieval. Subsequently, the sections were incubated at 37 ℃ for about 20 min with universal secondary antibodies and then stained with 3,3'-diaminobenzidine (DAB) for a duration of 3–10 min. ABP1 expression was considered positive if ≥5% of the cells exhibited positive staining.

Enzyme-linked immunosorbent assay (ELISA)

Urine ABP1 level was detected using ELISA.Utilizing an ELISA kit (MLBIO, Shanghai, China), we determined the number of antibody-coated microplates required based on the total number of urine and blood samples to be assayed. After the coating strips were removed, a marker pen was employed to demarcate the numbers of standard, sample, and blank wells on the antibody-coated microplates. If the kit had been stored at 4 ℃, it was allowed to thaw at room temperature prior to use.The standard solution was prepared according to the kit instructions. Then, 50 µL of the prepared standard solution was pipetted into the standard wells. After adding fresh urine (10 µL) or blood sample (10 µL), 40 µL of sample diluent was added. The blank wells contained no samples or reagents.Subsequently, 100 µL horseradish peroxidase (HRP)-conjugated anti-ABP1 antibody reagent was added to each standard or sample well, which was sealed with a plate sealer and then incubated in a 37 ℃ incubator for 1 hour. After the liquid in the reaction wells was discarded, blot the plate on absorbent paper to dry, then add washing solution into each well and let it stand for 30 seconds. Proceed to shake off the washing solution and blot the plate dry again on absorbent paper. Such a washing procedure was repeated five times. Subsequently, add 50 µL of chromogenic reagent A, followed by 50 µL of chromogenic reagent B, into each reaction well and incubate the plate at 37 ℃ for 15 minutes in the dark. Fifty microliters of stop solution were added to each reaction well. The optical density (OD) at 450 nm was measured for each well using a microplate reader. The exported data were plotted as standard curves using Excel, and the actual concentration of AOC1 was determined using the derived formula.

Experimental procedures of transwell cell migration assay

(I) PC3 (prostate cancer cell line) cells of the treatment group of AOC1 gene overexpression and the control group (untreated group) and transwell chambers were incubated at 37 ℃; (II) the cells were cultured to the logarithmic growth phase. The concentration was adjusted to 2×10³/mL; (III) adding medium containing 10% serum to the lower chamber, and cell suspension to the upper chamber. Continuing to incubate in the incubator for 24 hours; (IV) fixing cells with 4% paraformaldehyde for 30 minutes; (V) removing the chamber, dring the upper chamber fixative, with Giemsa staining solution for 15–30 minutes, then air-drying; (VI) cell counts of nine random field were taken under the microscope, and the results were statistically analyzed.

Statistical analysis

Statistical analyses were conducted using IBM SPSS software package (version 25.0). Chi-squared tests were performed with fourfold tables or R × C contingency tables, and Spearman’s rank correlation analysis was applied for correlation studies. Univariate survival analysis was conducted by employing the Kaplan-Meier method. Protein concentrations in blood and urine samples were analyzed using independent samples t-test. A P value of <0.05 was considered statistically significant.

Results

AOC1 expression in prostate cancer tissues

Immunohistochemistry indicated that the positive rate for AOC1 was 4.0% (4/101) in prostate cancer tissues, significantly lower than that (90.9%; 20/22) in normal prostate tissues (P<0.001) (Figure 1). AOC1 positivity in 4 prostate cancers was primarily focal expression, with rates of 5%, 5%, 5%, and a maximum of 30%. In normal prostate tissue, the situation of AOC1 positivity also lacked diffuse expression, with positive cells of 5–80%, an average number of positive cells of 39%, and median 40%. AOC1 expression in prostate cancer was positively correlated with postoperative recurrence and metastasis (P=0.004), and was related to invasion of the prostate capsule (P=0.04), and tended to be positively correlated with tumor node metastasis (TNM) stage and intravascular malignant thrombi, but showed no significant correlation with other clinicopathological features such as age and tumor necrosis (Table 1).The AOC1-positive subgroup demonstrated a significantly reduced cancer-specific survival (CSS) compared to the AOC1-negative subgroup (79.068±4.818 vs. 31.750±8.384 months; P<0.001) (Figure 2). The AOC1-positive subgroup demonstrated a significantly reduced progression-free survival (PFS) compared to the AOC1-negative subgroup (73.621±5.584 vs. 25.000±9.760 months; P=0.002).

Figure 1 Expression of AOC1 in prostate cancer and normal prostate tissues. (A1) H&E staining of normal prostate tissue. (A2) Expression of AOC1 in the normal prostate tissue from the same patient, with some glands exhibiting strong positivity. (B1) H&E staining of the junction between cancerous (black arrow) and normal prostate tissues. (B2) AOC1 is partially expressed in the adjacent normal tissue (red arrows) but not in the cancerous tissue (black arrow). (C1) H&E staining of prostate cancer tissue (black arrow) at low magnification, and red arrows show normal prostate tissue. (C2) In the same patient, AOC1 is not expressed in the prostate cancer tissue (black arrow), whereas partial expression is observed in the adjacent normal tissue (red arrows). (D1) H&E staining of prostate adenocarcinoma tissue with a Gleason grading 4. (D2) In the same patient, AOC1 is partially expressed in the prostate cancer tissue. A2, B2, C2, and D2: immunohistochemical staining performed with the EnVision polymer detection method. H&E, hematoxylin and eosin.

Figure 2 Effects of AOC1 expression in prostate adenocarcinoma on cancer-specific survival. Patients with AOC1-positive tumors have a shorter CSS (P<0.001). CSS, cancer-specific survival.

Including the expression of AOC1, age, stage, intravascular malignant thrombi, tumor necrosis, invasion of the prostate capsule and Gleason score, multivariate Cox regression analysis showed that the positive expression of AOC1 was the only prognostic factor of affecting CSS [P=0.005, hazard ratio (HR) 8.599, 95% confidence interval (CI): 1.921–34.496], or PFS (P=0.01, HR 5.444, 95% CI: 1.425–20.804).

AOC1 levels in blood and urine samples of prostate cancer patients

Preoperative AOC1 protein levels in the blood samples of prostate cancer patients did not significantly differ from levels three months postoperatively (P=0.41) (Figure 3). Similarly, no statistically significant difference was found in the urine AOC1 protein levels among the preoperative prostate cancer patients, those three months after operation of prostate cancers (P=0.43).

Figure 3 AOC1 protein levels in urine and blood samples. (A) The AOC1 protein level in urine. (B) The AOC1 protein level in blood. No statistically significant differences in AOC1 protein levels were observed between the prostate cancer group and prostate cancer 3 months post-surgery group. ns, not significant.

AOC1 expression in the urinary system and other male reproductive systems

To further understand the profile of AOC1 in surrounding tissues and to understand the possible sources of AOC1 in urine, we observed the expression of AOC1 in surrounding and adjacent tissues. Among the normal tubules, proximal convoluted tubule was found to have the highest AOC1 expression, with slightly reduced expression observed in the collecting tubules. In the present study, AOC1 showed certain expression in the seminal vesicle gland and epididymal epithelium, which was associated with cellular proliferation and basement membrane formation (Figure 4). Neither urothelium nor urothelial carcinoma had AOC1 expression (Table 2). However, AOC1 expression was detected in the cell membranes of urethral glands.

Figure 4 AOC1 expression in seminal vesicles and epididymal ducts. (A1) H&E staining of seminal vesicles from a patient. (A2) Partial expression of AOC1 in the seminal vesicles of the same patient (. (B1) H&E staining of the epididymal duct from one patient. (B2) In the same patient, partial expression of AOC1 was observed in the epididymal duct, with ciliary structures showing positive staining. A2 amd B2: immunohistochemical staining. H&E, hematoxylin and eosin.

Transwell cell migration assay of PC3 cells with AOC1 overexpression

The number of cell migration in the treatment group of AOC1 gene overexpression was significantly less than that in the control group (untreated group), indicating that this intervention significantly inhibited the migration ability of cells. The statistical results showed that the number of migrating cells decreased approximately 60% (Figure 5), with a significant difference (P<0.01), indicating that AOC1 gene had a strong inhibiting effect on PC3 cell migration.

Figure 5 Number of migrated PC3 cells were significantly reduced after AOC1 gene overexpression (P<0.01). Cells were stained with Giemsa. (A) The untreated group. (B) AOC1-overexpression group. HPF, high power field.

Discussion

AOC1 is a member of the copper/quinone oxidase family, encoding an 85-kDa protein that is typically expressed in placental and renal tissues. Locates on human chromosomes 7q34-q36, it may block sodium channels in the kidney (3). Human ABP/DAO corresponds to a polypeptide with 751 residues (4). Gene sequencing has revealed that a 2.4-kb mRNA was transcribed from two close origins, thus identifying the proximal promoter. Our subsequent study (data not shown) confirmed the robust expression of AOC1 in renal epithelial cells (particularly those on proximal convoluted tubule). Similarly, Verity et al. (5) demonstrated that the cDNA of rat ABP shared 83% homology with the equivalent region of the human peptide sequence, amplifying the same 311-nucleotide cDNA from the rat descending colon and kidney. ABP1 catalyzes the degradation of compounds including putrescine, histamine, spermine, and spermidine. Polyamine and its diamine precursor, putrescine, are essential for cell growth and proliferation in all organisms (1). These compounds are involved in allergic and immune responses, cell proliferation, tissue differentiation, carcinogenesis, and potential apoptosis. Lopes de Carvalho et al. (6) demonstrated that in mammals, copper-containing amine oxidase (CAO), encoded by four genes (AOC 1–4), catalyzes the oxidation of primary amines into aldehydes. CAOs can be categorized based on two specific residues, X1 and X2, within their active site motif: T/S-X1-X2-NYD (6).

In the present study, AOC1 was found to be robustly expressed in a subset of normal prostate glands, indicating a potential role in promoting the growth of normal prostate tissue. This expression pattern may be associated with the differentiation of prostate tissue, as not all glands exhibited AOC1 expression.

Our current study demonstrated that AOC1 expression was significantly lower in the prostate cancer tissue compared to normal prostate tissue, suggesting a deficiency in amine metabolism in most prostate cancers. The CSS of the AOC1-positive subgroup was significantly shortened. AOC1 expression in human colorectal cancer (CRC) tissues was correlated with a poorer prognosis, particularly in patients presenting with liver metastases (7). In hepatocellular carcinoma, high expression of AOC1 was also associated with poor prognosis (8). According to Ding et al., high expression of AOC1 was associated with enhanced proliferation, migration and invasion of tumor cells, which promoted tumor progression by activating the IL-6/JAK/STAT3 signaling pathway. However, if AOC1 expression is too low, it may affect certain key cellular metabolic or signaling processes, which in turn may lead to cellular dysfunction or increased apoptosis, thereby affecting patient survival (9). Functional analysis of cells showed that AOC1 gene knockdown inhibited the proliferation, invasion, and migration of human gastric cancer cell line (10). Similar prognoses were seen in our patients with prostate cancer. AOC1 expression was detected in tissues with a Gleason score of 4+3 or higher, and most were 9–10 scores (3/4), but not in those with a Gleason score of 6 or 3+4, suggesting a potential association with tumor grade. In our current research, there was no correlation between AOC1 positive rate and Gleason score, which may be related to fewer positive cases.

In our study, the positive expression of AOC1 was positively correlated with invading the capsule, and tended to be related to stage and intravascular thrombi, which suggested that AOC1-positive prostate cancer was more aggressive, similar to the study of Ding et al. (9). Combining with the literature and our prostate cancer research, it was suggested that positive expression of AOC1 in cancer cells was associated with poor prognosis which was related to tumor aggressiveness.

The results of our present study also indicated that there was no significant difference in the blood and urine concentrations of AOC1 between the prostate cancer group and the postoperative group.

Ding et al. (11) demonstrated that the expression of AOC1 in prostate cancer was inversely correlated with Gleason score, T stage, lymph node metastasis, and distant metastasis; in contrast, elevated AOC1 expression was significantly associated with reduced proliferation and migration of prostate cancer cells both in vitro and in vivo. Wei et al. (12) discovered an inverse correlation between AOC1 expression and the risk of biochemical recurrence in prostate cancer. Similar results were also observed in our PC3 cells. After the overexpression of AOC1, the migration ability of PC3 cells was significantly reduced, which suggested that overexpression of AOC1 inhibited the growth of PC3 cells of prostate cancer. After AOC1 knockdown in PC3 cells, wild-type p53 expression was decreased, while cell cycle-related proteins and genes were upregulated (not shown in the results), thereby resulted in increased cell of proliferation. However, in our present study, 3 out of 4 patients with AOC1 expression succumbed to the disease, suggesting that AOC1 might be a suitable target for therapeutic intervention in prostate cancer. We also found that most of AOC1 was not expressed, and AOC1 expression was positively correlated with recurrence and metastasis.

Why did the opposite results occur? There are several reasons as follows:

• Among our 4 cases, all were from patients with high AR expression and were androgen-dependent. However, our cell experiment used PC3 cells (which are non-androgen-dependent), and the signaling pathways differ between androgen-dependent and non-androgen-dependent (cell/tumor) models.
• Furthermore, in AOC1-positive cases, the proportion of positive cells was low, with most cases accounting for approximately 5% and only one case reaching around 30%, meaning they did not constitute the main component of the tumor.
• In 1 positive case, a small number of CK20-positive cells were detected, which suggested intestinal differentiation.
• In 3 cases, the staining was localized to the Gleason grade 4 area, and the stained area contained small glandular lumens, which also suggested that it was a secretory protein, while the remaining 1 case showed the stainning of AOC1 in the Gleason grade 5 region. The stained cells have abundant cytoplasm, and 2 cases showed that the cells were lightly stained or translucent.
• In our study, there were 4 positive cases, 3 of which were stage IV and 1 was stage III. We speculated that AOC1 played a tumor-suppressing role in the early stage of prostate cancer, and its loss led to the occurrence of prostate cancer. In the middle to advanced stages of prostate cancer, the expression of AOC1 promoted the progression of prostate cancer.

AOC1 was not expressed in the vast majority of prostate cancer tissues but was expressed in some normal glandular tissues, suggesting that most prostate cancers lacked AOC1-related signaling pathways. However, in certain cases, cancer tissues from patients with focal positive AOC1 expression exhibited a function of promoting cancer cell invasion, which implies the involvement of multiple signaling pathways. Combined with the established pro-carcinogenic role of AOC1 in gastrointestinal cancers and hepatocellular carcinoma (8-10), it is hypothesized that AOC1 may exert bidirectional functions in prostate tissue: it may generally inhibit prostate cancer cell proliferation, while under specific microenvironmental conditions, AOC1 expression can also promote the invasion of cancer cells.

Conclusions

AOC1 expression is low in prostate cancer, and the positive expression is associated with a poor prognosis. In normal prostate tissue, AOC1 is expressed in some normal glands, which may be associated with the growth and differentiation of the normal prostate. In the future, further research is needed to confirm the findings.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-258/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-258/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-258/prf

Funding: This work was supported by the Clinical Medical Research Transformation Special Project of Anhui Province (No. 202204295107020051); and the Excellent Scientific Research and Innovation Team of Anhui Universities (No. 2024AH010021); and the Co-construction Project of the Bengbu Municipal Health Commission (No. BBWK2024A105).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-258/coif). All authors report funding support from the Clinical Medical Research Transformation Special Project of Anhui Province (No. 202204295107020051); and the Excellent Scientific Research and Innovation Team of Anhui Universities (No. 2024AH010021); and the Co-construction Project of the Bengbu Municipal Health Commission (No. BBWK2024A105). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by ethics committee of the Third People’s Hospital of Bengbu (ethical No. 2023-01). Individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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