RAB27A, RAB27B and VPS36 are downregulated in advanced prostate cancer and show functional relevance in prostate cancer cells

Abstract

Paracrine and long-range signaling via extracellular vesicles, such as exosomes and microvesicles, is deemed crucial for tumorigenesis, invasion and spread of solid tumors. The ESCRT machinery (endosomal sorting complexes required for transport) and Rab-proteins act as key players in vesicular trafficking and secretion. Yet, their role in prostate cancer (PCa) is unknown. Therefore, this study aimed to elucidate the relevance of these components in PCa. In silico reanalysis of genes with known involvement in vesicular trafficking and secretion in an existing microarray dataset revealed low expression of RAB27A, RAB27B and VPS36 to be predictive for reduced BCR-free survival in patients with localized PCa (p=0.033, 0.025 and 0.005). In the same microarray dataset underexpression of RAB27A, RAB27B and VPS36 was seen in distant metastases (p<0.001; p=0.003; p<0.001). This was consistent in two further microarray datasets. qRT-PCR-validation in two independent cohorts of PCa specimens (n=90) showed low expression of VPS36 in PCa tissue (p=0.023), especially in castration-resistant tumors (p=0.002). In all five datasets there were significant correlations between the expression of at least two of the candidates. Upon knockdown of VPS36 an increase of RAB27A and RAB27B expression, but not vice versa, was observed in both prostate and breast cancer cells (PC3, MDA-MB‑231). In PC3 cell knockdown of RAB27B and VPS36 dramatically reduced colony formation (-52.2%, p<0.001; -71.1%, p<0.001) and, controversial to reports in other tumor entities, increased the release of extracellular particles (+25.3%, p=0.014; +45.6%, p<0.001). Taken together RAB27A, RAB27B and VPS36 are frequently underexpressed in advanced PCa and are inversely correlated with PCa outcome. There seems to be a close relationship in the expression of RAB27A, RAB27B and VPS36, with RAB27A and RAB27B being dependent on VPS36. Changes in colony formation and particle release upon RNAi indicate an involvement in paracrine cell-cell communication.

Current knowledge on exosome biogenesis and release

“…Moreover, Rab7 and Rab27b, but not Rab27a, have been shown to regulate secretion of exosomal miR-143 from HUVEC cells [ 89 ]. Contrary to previous studies, a recent report showed that knockdown of Rab27b increased particle release from PC-3 cells [ 90 ]. However, in this study exosomes were not isolated and the conditioned media was measured by NTA directly after 12,000× g centrifugation.…”

Rab27A overexpression promotes bladder cancer proliferation and chemoresistance through regulation of NF-κB signaling

“…High Rab27A expression indicates favorable prognosis in colorectal cancer [ 12 ]. RAB27A and RAB27B are also downregulated in advanced prostate cancer [ 13 ]. Thus the expression pattern and role of Rab27A is tissue specific.…”

The EEF1A2 gene expression as risk predictor in localized prostate cancer

“…RNA-extraction was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. cDNA-Synthesis was performed as described previously [ 19 ]: in brief 40 μl of diluted RNA were mixed with 4 μl of 5 mg/ml pdN6 random primers, 4 μl of 10 mM dNTP Mix, 16 μl of 5× M-MLV buffer, 8 μl of 0.1 M RNase inhibitor, 4 μl of 0.1 M DTT and 4 μl of M-MLV reverse transcriptase (all from Roche Diagnostics, Basel, Switzerland). After an incubation for 2 h at 37 °C and a deactivation step of 5 min at 65 °C, cDNA was directly used for qRT-PCR or stored at −20 °C.…”