Abstract
Epithelial-mesenchymal transition (EMT) is a critical early step in cancer metastasis and a complex process that involves multiple factors. In this study, we used proteomics approaches to investigate the secreted proteins (secretome) of paired human androgen-repressed prostate cancer (ARCaP) cell lines, representing the epithelial (ARCaP-E) and mesenchymal (ARCaP-M) phenotypes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses showed high levels of proteins involved in bone remodeling and extracellular matrix degradation in the ARCaP-M cells, consistent with the bone metastasis phenotype. Furthermore, LC-MS/MS showed a significantly higher level of the serine protease granzyme B (GZMB) in ARCaP-M conditioned media (CM) compared to that of ARCaP-E. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to detect mRNA and Western blot to detect protein expression, we further demonstrated that the GZMB gene was expressed by ARCaP-M and the protein was secreted extracellularly. ARCaP-M cells with GZMB gene knockdown using small interfering RNA (siRNA) have markedly reduced invasiveness as demonstrated by the Matrigel invasion assay in comparison with the scrambled siRNA negative control. This study reports that GZMB secretion by mesenchymal-like androgen-repressed human prostate cancer cells promotes invasion, suggesting a possible extracellular role for GZMB in addition to its classic role in immune cell-mediated cytotoxicity.
Highlights
Prostate cancer is the most common cancer among men in the United States, aside from nonmelanoma skin cancer, according to the Centers for Disease Control and Prevention (CDC)
Enriched processes in ARCaP-M secretome were involved in proteolysis and extracellular matrix (ECM) disassembly (S1 Table)
The upregulated ARCaP-M proteins involved in these processes include the aminopeptidases aminopeptidase N (ANPEP) and leucyl-cystinyl aminopeptidase (LNPEP) (6.8 and 5.7-fold, respectively), granzyme B (GZMB; 800-fold), and matrix metalloproteinase 1 (MMP1; 2.4-fold)
Summary
Prostate cancer is the most common cancer among men in the United States, aside from nonmelanoma skin cancer, according to the Centers for Disease Control and Prevention (CDC). Two-thirds of cancer-related deaths in the US involve bone metastasis and prostate tumors in particular are prone to disseminate to the bone [1]. Despite the recent advances in clinical trials, cancer metastasis still accounts for the majority of cancer deaths and metastatic prostate cancer remains an incurable disease [2,3,4]. Granzyme B secretion by ARCaP-M decision to publish, or preparation of the manuscript
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