Background:Novel biomarkers for prostate cancer (PC) are urgently needed. This study investigates the expression, epigenetic regulation, and prognostic potential of ANPEP in PC.Methods:Aminopeptidase N (APN; encoded by ANPEP) expression was analysed by immunohistochemistry using tissue microarrays representing 267 radical prostatectomy (RP) and 111 conservatively treated (CT) PC patients. Clinical end points were recurrence-free survival (RFS) and cancer-specific survival (CSS), respectively. The ANPEP promoter methylation levels were determined by bisulphite sequencing or MethyLight analysis in 278 nonmalignant and PC tissue samples, and in cell lines.Results:The APN expression was significantly downregulated in PC compared with nonmalignant prostate tissue samples. Aberrant promoter hypermethylation was frequently observed in PC tissue samples, and 5-aza-2′-deoxycytidine induced ANPEP expression in three hypermethylated prostate cell lines, suggesting epigenetic silencing. Negative APN immunoreactivity was significantly associated with short RFS and short CSS in the RP and CT cohort, respectively, independently of routine clinicopathological predictors. Combining APN with a known angiogenesis marker (vascular endothelial growth factor or microvessel density) improved risk prediction significantly in both cohorts.Conclusion:Our results suggest negative APN immunoreactivity as a new independent adverse prognostic factor for patients with clinically localised PC and, furthermore, that epigenetic mechanisms are involved in silencing of ANPEP in PC.
This study investigates the expression and biomarker potential of zinc finger protein 132 (ZNF132) in prostate cancer (PC) by transcriptional profiling and immunohistochemical analysis of tissue microarrays, including tumor specimens from 615 radical prostatectomy (RP) patients and 199 conservatively treated patients. Primary clinical endpoints were time to PSA recurrence and cancer-specific death, respectively. Compared to normal prostate epithelial cells from men without PC, ZNF132 transcript levels were significantly reduced in PC cells from patients with localized PC and further downregulated in metastatic PC. Likewise, ZNF132 protein expression was significantly lower in primary tumors from patients with metastatic compared to localized PC and further reduced in castrate-refractory PC, indicating that ZNF132 downregulation correlates with disease progression. Reduced ZNF132 immunoreactivity was significantly associated with high Gleason score and advanced T stage in both PC patient cohorts. By univariate analysis, no/weak ZNF132 staining was a significant adverse predictor of PSA recurrence after RP (p 5 0.024) and cancer-specific death following conservative treatment (p 5 0.009). In multivariate models, however, ZNF132 did not add significant independent value to established prognostic factors. Finally, bisulfite sequencing revealed frequent promoter hypermethylation of ZNF132 in both PC cell lines and PC tissue samples, indicating that ZNF132 is epigenetically silenced in PC. In summary, our results show that downregulation of ZNF132 is associated with aggressive PC and furthermore identify ZNF132 as a new candidate methylation marker for PC.
Prostate cancer (PC) is a common malignancy and a leading cause of cancer-associated mortality among males in Western countries. Novel diagnostic and prognostic biomarkers for PC are urgently needed to prevent overdiagnosis and overtreatment of nonaggressive tumors. In this study, we investigated the biomarker potential of ANPEP in PC. ANPEP encodes a membrane-bound zinc-dependent protease termed aminopeptidase N (APN), which belongs to a group of widely expressed ectopeptidases. Expression of APN protein was determined by immunohistochemical analysis of tissue microarrays, including PC specimens from 267 radical prostatectomy (RP) patients and 199 conservatively treated (CT) patients as well as several nonmalignant prostate and metastatic PC samples. Clinical endpoints were recurrence-free survival (RFS) and cancer-specific survival (CSS), respectively. ANPEP promoter methylation was analyzed in parallel by quantitative methylation-specific PCR (qMSP/MethyLight) in approximately 250 nonmalignant and PC tissue samples. We found that APN protein expression was significantly lower in localized PC and further reduced in metastatic PC, compared to nonmalignant prostate tissue samples. Aberrant ANPEP promoter hypermethylation was observed in approximately half of all PC samples investigated. Methylation levels correlated inversely with APN immunoreactivity, suggesting that ANPEP is epigenetically silenced in PC. By multivariate analysis, negative APN immunoreactivity was significantly associated with short RFS in the RP cohort (HR=0.24; p=0.002) and with short CSS in the CT cohort (HR=0.23; p=0.049), in both cases independently of routine predictors such as Gleason score, T stage and PSA. Combining APN with a known angiogenesis marker (vascular endothelial growth factor (VEGF) immunoreactivity or microvessel density (MVD)) in a simple two-marker model improved risk prediction significantly in both cohorts. ANPEP promoter methylation did not show significant prognostic value in the RP cohort (p=0.09) and was not investigated in the CT cohort. In conclusion, our results indicate that a simple immunohistochemical test for APN may add significant prognostic value to currently used routine predictors for PC outcome. Furthermore, our findings suggest that it may be favorable to use APN in combination with an established angiogenesis marker to improve risk stratification and separate patients with clinically localized PC into low, intermediate and high risk subgroups in order to guide treatment decisions. Finally, we have identified ANPEP as a new common target for promoter hypermethylation in PC, indicating that epigenetic mechanisms contribute to downregulation of ANPEP in PC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3636. doi:1538-7445.AM2012-3636
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