Carboxypeptidase-D is elevated in prostate cancer and its anti-apoptotic activity is abolished by combined androgen and prolactin receptor targeting

BACKGROUND Carboxypeptidase-D (CPD) cleaves C-terminal arginine for nitric oxide (NO) production. CPD and NO levels are upregulated by testosterone (T) and prolactin (PRL) to promote survival of prostate cancer (pCa) cells. This study evaluated CPD immunostainingand T/PRL regulation of CPD and NO levels in benignand malignant prostate tissues/cells to determine the role of CPD in pCa. METHODS Immunohistochemistry (IHC) and tissue microarrays(TMA) were used to determine CPD immunostainingin prostate specimens. QPCR and immunoblotting were used to quantify CPD mRNA/protein expression in prostate cells. NO production was measured using 4,5-diaminofluorescein diacetate assay. RESULTS CPD staining increased from 8.9 ± 3.8% (Mean ± SEM, n = 15) of benignepithelial cell area to 30.9 ± 2.9% (n = 30) of tumor cell area in one set of TMAs (P = 0.0008) and from 5.9 ± 0.9% (n = 45) of benignepithelial cell area to 18.8 ± 1.9% (n = 55) of tumor area in another (P < 0.0001). IHC of prostate tissues (≥50 mm2) confirmed increased CPD staining, from 13.1 ± 2.9% in benign(n = 16) to 29.5 ± 4.4% in pCa (n = 31, P = 0.0095). T and/or PRL increased CPD expression in several pCa but not benigncell lines. T and PRL acted synergistically to increase NO production, which was abolished only when receptor antagonists flutamideand Δ1–9-G129R-hPRL were used together. CONCLUSIONS CPD immunostainingand T/PRL-stimulated CPD expression were higher in pCa than benigntissues/cells. Elevated CPD increased NO production, which was abolished when both AR and PRLR were inhibited. Our study implicates a critical role for the T/PRL-stimulated CPD-Arg-NO pathway in pCa progression, and suggests that AR+PRLR inhibition is a more effective treatment for pCa. Prostate 74:732–742, 2014. © 2014 Wiley Periodicals, Inc.