MicroRNA (miRNA) expression profiles were generated from prostate epithelial subpopulations enriched from patient-derived benign prostatic hyperplasia (n = 5), Gleason 7 treatment-naive prostate cancer (PCa) (n = 5), and castration-resistant PCa (CRPC) (n = 3). Microarray expression was validated in an independent patient cohort (n = 10). Principal component analysis showed that miRNA expression is clustered by epithelial cell phenotype, regardless of pathologic status. We also discovered concordance between the miRNA expression profiles of unfractionated epithelial cells from CRPCs, human embryonic stem cells (SCs), and prostate epithelial SCs (both benign and malignant). MiR-548c-3p was chosen as a candidate miRNA from this group to explore its usefulness as a CRPC biomarker and/or therapeutic target. Overexpression of miR-548c-3p was confirmed in SCs (fivefold, p < 0.05) and in unfractionated CRPCs (1.8-fold, p < 0.05). Enforced overexpression of miR-548c-3p in differentiated cells induced stemlike properties (p < 0.01) and radioresistance (p < 0.01). Reanalyses of published studies further revealed that miR-548c-3p is significantly overexpressed in CRPC (p < 0.05) and is associated with poor recurrence-free survival (p < 0.05), suggesting that miR-548c-3p is a functional biomarker for PCa aggressiveness. Our results validate the prognostic and therapeutic relevance of miRNAs for PCa management while demonstrating that resolving cell-type and differentiation-specific differences is essential to obtain clinically relevant miRNA expression profiles.
We report microRNA (miRNA) expression profiles of epithelial cell fractions from the human prostate, including stem cells. miR-548c-3p was revealed as a functional biomarker for prostate cancer progression. The evaluation of miR-548c-3p in a larger patient cohort should yield information on its clinical usefulness.
Keywords: MicroRNA, Stem cells, Biomarker, Castration-resistant prostate cancer.
The identification of improved biomarkers and treatment strategies for castration-resistant prostate cancer (CRPC) remains a priority in prostate cancer (PCa) research. Since their discovery, microRNAs (miRNAs) have shown promise in both fields . Indeed, miRNA-focused research has yielded >2000 patents and several clinical trials for cancer management ; however, clinical translation of miRNA as a PCa biomarker and/or as a novel therapeutic target remains more limited. This situation is perhaps because of the considerable heterogeneity and discrepancies in PCa miRNA expression profiles  and . Most miRNA expression patterns are cell type–specific, but they also change with cellular differentiation status, even in cancer . We set out to investigate whether the failure to resolve cell type–specific and differentiation-specific differences has contributed to the significant variations in published PCa miRNA profiles.
We have previously shown that a CD133+α2β1hi subpopulation enriched from benign and cancerous prostate tissue expresses high levels of CD44 and exhibits stem cell (SC) properties  and . Genome-wide miRNA expression analysis was performed on patient-derived stemlike cells (SC-CD133+α2β1hi), transit-amplifying cells (TA-CD133−α2β1hi), and committed basal (CB) cells (CB-CD133−α2β1lo) enriched from briefly cultured primary prostate epithelial cells (Fig. 1a, Supplement, Supplementary Table 1)  and . The validity of miRNA expression data was confirmed by examining the expression patterns of 11 randomly selected miRNAs using quantitative reverse transcription polymerase chain reaction analysis (Supplementary Fig. 1). Subsequent principal component analysis clearly demonstrated that each subpopulation, regardless of its pathologic status, had a distinct miRNA expression profile (Fig. 1a). The magnitude and the extent of differential miRNA expression in SCs compared with CB cells were also significantly higher than in benign prostatic hyperplasia (BPH) versus PCa or in BPH versus CRPC, indicating that the differentiation stage of a prostate epithelial cell is the primary determinant of its miRNA expression profile.
Cell subtype, rather than pathologic status, is a primary determinant of microRNA (miRNA) expression. (a) A schematic of human prostate epithelial hierarchy (left) showing a stemlike cell with a basal phenotype subsequently differentiating into luminal cells by way of committed basal (CB) cells. The subpopulations were enriched from normal human prostate epithelial cells, benign prostatic hyperplasia (BPH), and cancers (high Gleason grade, treatment-naive prostate cancer [PCa], and castration-resistant PCa [CRPC]). Principal component analysis was performed on miRNA microarray profiles of cultured stem cells (SCs) and CB cells at passage 2 (right). (b) Comparison of miRNA expression profiles for unfractionated CRPC tissue (vs BPH)  and prostate SC (vs CB). (c) Kaplan-Meier curve for PCa patient survival with differential miR-548c-3p expression using Taylor et al. . (d) Colony-forming efficiency of miR-548c-3p transfected CB cells (n = 3 for BPH, n = 3 for PCa; each sample in triplicate). (e) Fluorescence-activated cell sorting analysis for CD49b (integrin β2) and CD49f (integrin β6) expression performed on CB cells transfected with either control or miR-548c-3p for 3 d (n = 3 for BPH, n = 3 for PCa; each sample in triplicate). (f) Live cell count of miR-548c-3p transfected CB cells 48 h after exposure to 5-Gy radiation (n = 3 for BPH, n = 3 for PCa; each sample in triplicate). (g) Quantitative reverse transcription polymerase chain reaction analysis for miR-548c-3p expression in epithelial cells enriched from freshly disaggregated uncultured BPH tissue (n = 3), PCa Gleason grade 7 tissue (n = 5), and CRPC tissue (n = 3). Each sample was assessed in triplicate. Data are expressed as mean plus or minus standard deviation. *p < 0.05 (student t test); **p < 0.01 (student t test); ***p < 0.001 (student t test).
BPH = benign prostatic hyperplasia; CFE = colony-forming efficiency; CRPC = castration-resistant prostate cancer; miRNA = microRNA; PCa = prostate cancer; PREC = prostate epithelial cell.
Further examination of the miRNA expression profiles led to the following interpretations. First, a prostate epithelial SC signature is conserved in BPH, PCa, and CRPC (Supplementary Table 2), suggesting that miRNAs may primarily regulate core SC properties (self-renewal, prolonged proliferation, and differentiation capability), which are common for the SC phenotype regardless of its pathologic status. Second, conserved prostate SC miRNA signatures share their miRNA expression pattern with human embryonic SCs (hESCs) , for example, higher expression of miR-302/372 families and suppression of the let-7 family (Table 1). Third, there is an overlap of approximately 60% between the miRNA expression profiles of SCs and those of previously published unfractionated CRPCs  (Fig. 1b). Several of these shared miRNAs potentially regulate key SC and cancer-associated proteins; for example, miRNAs potentially regulating c-MYC, KLF4, NANOG, and EZH2 are all suppressed in SCs and CRPCs. Fourth, it is possible to distinguish between PCa–cancer stemlike cell (CSC), CRPC-CSC, and normal SC signatures, as well as signatures from their respective differentiated progeny (Table 1). Fifth, composite PCa and CRPC miRNA signatures identified in this paper contain several previously known onco-miRs and tumour suppressor miRNAs (eg, miR-629 and miR-203) (Supplementary Table 3).
MicroRNA signatures of conserved stem cells, prostate cancer stemlike cells, and castration-resistant prostate cancer stemlike cells
|SC signature||Specific PCa CSC signature||Specific CRPC CSC signature|
* Indicates the non-predominant product of a specific miRNA locus.
CRPC = castration-resistant prostate cancer; CSC = cancer stemlike cell; PCa = prostate cancer; SC = stem cell.
Our miRNA expression analysis of patient-derived prostate epithelial subpopulations has therefore identified several novel PCa-CSC–specific and CRPC-CSC–specific miRNA candidates. The analyses also identified previously well-established miRNAs associated with PCa (eg, consistent suppression of miR-299–5p, which is downregulated in metastatic cell lines compared with normal prostate epithelial cells) , CRPC (eg, miR-521, whose inhibition in LNCaP cells enabled acquisition of a radioresistant phenotype) , and CSCs (eg, miR-708, whose suppression allows upregulation of CD44 and Akt in prostrate xenograft–derived cells) . These correlations also imply that the hESC maintenance program is partly conserved in adult human prostate epithelial SCs at the miRNA level, which is in turn hijacked by the malignant cells in CRPCs.
To illustrate the relevance of our data set, we decided to investigate the role of miR-548c-3p during prostate epithelial differentiation and carcinogenesis (based on criteria described in Supplementary Fig. 2). This miRNA is overexpressed approximately fivefold in prostate epithelial SCs compared with CBCs (Supplementary Fig. 3), and its overexpression has been associated with poor survival of PCa patients  (p = 0.0389, log-rank test) (Fig. 1c). Overexpression of miR-548c-3p in CB cells (Supplementary Fig. 3) resulted in dedifferentiation to a more stemlike phenotype as (1) the colony-forming efficiency increased by approximately 75%, which is a commonly used indicator for SC self-renewal (Fig. 1d); (2) expression of the prostate epithelial stem/progenitor cell proteins CD49b (integrin β2) and CD49f (integrin β6) increased by 50–80% (Fig. 1e); (3) there was an increase in mRNA expression of multiple SC-specific genes with a concomitant reduction in CB cell–specific genes (Supplementary Fig. 3); and (4) CB cells became radioresistant, as an increase in live cell count of approximately 25% was noted 48 h after exposure to 5-Gy radiation (Fig. 1f).
Analyses of potential miR-548c-3p targets (Supplementary Fig. 4), together with our functional data, implicate miR-548c-3p in SC maintenance and cell cycle regulation. An independent study has shown that over-expression of miR-548c-3p decreased doxorubicin-induced DNA damage in cervical cancer cell line (HeLa cells through inhibition of topoisomerase (DNA) II alpha 170kDa (TOP2A) . A reduction in DNA damage, an increase in cell proliferation, and the acquisition of stemlike properties have all been reported in CRPCs. We indeed found miR-548c-3p to be significantly upregulated in uncultured CRPC-derived epithelial cells compared with BPH-derived epithelial cells (Fig. 1g), which eliminated the possibility of cell culture artifact. Others have further demonstrated that serum obtained from CRPC patients contained 2.8-fold higher miR-548c-3p levels compared with serum derived from low-risk PCa patients . These results attest to the importance of miR-548c-3p as a strong diagnostic and prognostic candidate to improve CRPC patient management. Clinical validation in a larger patient cohort is now necessary to establish therapeutic relevance.
The molecular programs that drive epithelial SC lineage commitment toward a differentiated phenotype (in an adult human prostate) remain unexplained. This analysis provides the first comprehensive input toward enabling an understanding of key miRNA expression changes during prostate epithelial differentiation. The overlap between the miRNA expression patterns of hESCs, prostate epithelial SCs, and unfractionated CRPCs clearly illustrates that embryonic signalling machinery is activated in the terminal stages of PCa.
In conclusion, our investigation identifies the failure to resolve cell subtype–specific miRNA expression differences as one of the reasons behind previously observed heterogeneous miRNA expression profiles of unfractionated prostate tumours. The data also provide novel and clinically relevant miRNA-based therapeutic candidates, including miR-548c-3p, for the management of CRPCs and CSCs. Further integration of this miRNA data set with mRNA data obtained from similarly fractioned subpopulations from PCa and CRPC should now enable the resolution of multidimensional transcriptional interrelationships in human prostate epithelium.
Author contributions: Norman J. Maitland had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Rane, Pellacani, Visakorpi, Maitland.
Acquisition of data: Rane, Scaravilli, Ylipää.
Analysis and interpretation of data: Rane, Ylipää, Pellacani, Nykter, Visakorpi, Maitland.
Drafting of the manuscript: Rane, Maitland.
Critical revision of the manuscript for important intellectual content: Rane, Scaravilli, Ylipää, Pellacani, Nykter, Collins, Visakorpi, Maitland.
Statistical analysis: Rane, Ylipää.
Obtaining funding: Visakorpi, Maitland.
Administrative, technical, or material support: Scaravilli, Mann, Simms, Nykter, Maitland.
Supervision: Nykter, Visakorpi, Maitland.
Other (specify): None.
Financial disclosures: Norman J. Maitland certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None.
Funding/Support and role of the sponsor: The work was funded by a PRO-NEST Marie-Curie Grant (Jayant K. Rane and Mauro Scaravilli); the Finnish Funding Agency for Technology and Innovation Finland Distinguished Professor programme and Academy of Finland: project no. 132877 (Antti Ylipää and Matti Nykter); and Yorkshire Cancer Research (Davide Pellacani, Vincent M. Mann, Anne T. Collins, and Norman J. Maitland).
Acknowledgment statement: We would like to thank all the patients and urology surgeons L. Coombes, G. Cooksey, and J. Hetherington (Castle Hill Hospital, Cottingham, UK).
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a YCR Cancer Research Unit, Department of Biology, University of York, York, North Yorkshire, UK
b University of Tampere and Tampere University Hospital, BioMediTech, Molecular Biology of Prostate Cancer Group, Tampere, Finland
c Department of Signal Processing, Tampere University of Technology, Tampere, Finland
d Terry Fox Laboratory, Eaves Lab, BC Cancer Research Centre, Vancouver, BC, Canada
e Hull York Medical School, University of Hull, Hull, East Yorkshire, UK
f Department of Urology, Castle Hill Hospital, Cottingham, East Yorkshire, UK
Corresponding author. Tel. +44 0 1904 328700; Fax: +44 0 1904 328710.
© 2014 European Association of Urology, Published by Elsevier B.V.