One out of six men will be diagnosed with prostate cancer during their lifetime (1
). However, using current risk stratification strategies it is still difficult to separate patients with rapidly progressing tumors, which require aggressive treatment, from those with indolent tumors. The result is over treatment, leading to undesirable side affects for a large population of men and unnecessary consequential healthcare costs (2
). Therefore, it is essential that new biomarkers are uncovered that better predict outcome of the disease and hence enable better individual treatment decisions.
Cancer biomarkers provide a potentially powerful tool in the diagnosis / prognosis of cancer patients. In prostate cancer, the commonly used biomarker is PSA, a protein antigen specific to prostate tissue, which is measured in the blood and elevated in prostate cancer. Unfortunately, PSA alone is a poor predictor of disease outcome (3
). Recently, microRNAs (miRNAs) have been found in serum and have been proposed as potential biomarkers of normal physiology and disease (4
). MiRNAs are short (18
nucleotides), non-coding RNAs, which regulate gene expression post-transcriptionally by both destabilizing messenger RNAs (mRNA) and inhibiting their translation. Canonical miRNAs derive from longer polymerase II transcripts, called pri-miRNAs. A complex consisting of the proteins DGCR8 and Drosha process the pri-miRNAs to pre-miRNAs, which are then exported to the cytoplasm and cleaved by the protein Dicer to mature miRNAs. Exceptions to this processing include non-canonical miRNAs that bypass DGCR8/Drosha, while still being processed by Dicer. Knockout models of Dgcr8
have been developed that remove only canonical miRNAs or both canonical and non-canonical miRNAs respectively (5
For prostate cancer tissue there have been very few studies that compared miRNA expression levels in more than 10 benign and malignant samples (8
). Noticeably, there is no overlapping subset between the down- or upregulated miRNAs (12
). Mitchel et al. were the first to show a correlation between miRNAs found in plasma and the presence of prostate cancer. They demonstrated that miRNAs originating from human prostate cancer xenografts can enter the circulation, be measured in plasma and robustly distinguish xenografted mice from controls. Furthermore, they found that serum levels of miR-141 distinguished patients with advanced prostate cancer from healthy controls (14
). Lodes et al screened 21 sera from 5 different cancer entities (colon, ovarian, breast, lung and prostate). In total 6 prostate cancer patient samples were studied, from which 5 were from patients in advanced prostate cancer stages (3 and 4) and only one sample was from a non-advanced stage. Four of the total 6 prostate cancer patients in this study had received systemic chemotherapy (Taxotere or Lupron with/without the bisphosphonate Zometa). Therefore it remains unclear whether the altered serum miRNA levels are a result of the chemotherapy or reflect the actual advanced cancerous disease (15
To establish a method for identifying miRNA signatures that could provide diagnostic and prognostic information for prostate cancer patients prior to any treatment, we developed a novel multiplex qRT-PCR method and tested 4 sets of patients (12 patients in each set) with differing risk stratifying CAPRA scores. Due to the inconsistency of previous profiling experiments (12
) our multiplex qRT-PCR technique was validated using wild type mESC, Dgcr8
knockout and Dicer
knockout mESC as negative controls, showing much improved accuracy relative to currently published protocols. Using this technique on a microfluidic platform we screened patient serum samples for the expression level of 384 miRNAs and uncovered miRNA signatures that diagnosed prostate cancer samples and correlated, but did not exactly match patient CAPRA scores, suggesting that these signatures may have the potential to separate patients within the risk groups and provide additional prognostic information. Many of the miRNAs found to correlate with groupings have been shown to have oncogenic or tumor suppressive functions in different cancer contexts and, therefore, altered miRNA levels in the serum may reflect functional roles within the tumors.