Background
Currently, the most frequently used methods for detecting prostate cancer are a digital rectal exam and a blood test to determine levels of prostate-specific antigen (PSA) produced by the prostate gland [
1]. However, these diagnostic tools can lack the sensitivity required to detect very early prostate lesions [
2]. Furthermore, PSA levels can increase for reasons unrelated to cancer or not increase when cancer is present [
2]. If a prostate cancer is suspected, prostate biopsies are performed. However, prostate biopsies are invasive, and can lead to false-negatives and repeat biopsies, as they do not sample the entire prostate. Recent developments in prostate cancer detection include measuring the non-coding RNA prostate cancer antigen 3 (
PCA3) and transmembrane protease, serine 2 (
TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) (
ERG) gene fusion in urine to identify patients requiring repeat biopsies despite an initial negative biopsy [
3‐
5]. However, there is a clear need to identify novel biomarkers for diagnostic purposes that are sensitive and specific to prostate cancer.
Epigenetic patterns are known to be altered in several different cancer types, including prostate cancer, and signatures of DNA methylation may serve as potential diagnostic or prognostic biomarkers [
6]. Cancer-derived, methylated DNA has been identified and purified from both patient serum and urine, making it a promising option for a non-invasive biomarker [
7]. Previous studies investigating DNA methylation patterns at select genomic loci in prostate cancer resulted in discoveries of epigenetic differences between prostate cancer tissue and benign-adjacent prostate in genes such as glutathione s-transferase 1 (
GSTP1), Ras association domain family member 1 (
RASSF1), and adenomatous polyposis coli (
APC), among others [
8‐
10]. Recently, there have been studies using global approaches in prostate cancer that have identified DNA methylation alterations in malignant prostate tissue, including a previous study from our group [
11‐
17]. We sought to expand upon our previous discoveries by performing genome-wide measurements of DNA methylation in 73 clinically annotated fresh-frozen prostate cancers and 63 benign-adjacent prostate tissues using the Illumina Infinium HumanMethylation450 BeadChip array, which offers greater genomic coverage compared to the Methyl27 array that we previously used [
11]. We present here novel DNA methylation-based diagnostic models, and discuss transcription factors whose binding sites are enriched in regions of differential methylation in prostate cancer.
Discussion
Shifts in epigenetics play a large role in cancer formation and maintenance, and DNA methylation is a stable modification that can be detected non-invasively in fluids such as urine, blood and saliva. For these reasons, DNA methylation is an attractive cancer biomarker candidate. In our study, we identified a large number of CpG loci with statistically significant DNA methylation levels between our cohort of prostate cancer tissues and the adjacent, unaffected prostate tissues. More than half of the significant CpGs were found to be hypermethylated in the prostate tumor tissues. Our previous work strongly suggests that these methylation changes are the result of dysregulation of the DNA methyltransferases DNMT3A2 and DNMT3B [
11].
Global DNA methylation changes implicate genes associated with the stroma and tumor microenvironment as being enriched targets for methylation changes. We observed an overwhelming signature of glycosaminoglycan (GAG) metabolism in the regulatory regions of transcripts with higher methylation in malignant tissues. GAGs are long polysaccharides that have both structural and signaling roles within the extracellular matrix and cellular membranes and have a documented role in many cancers [
33]. In prostate cancer, altered expression of GAGs has been observed in early stage prostate cancer and correlates with malignant progression. A large body of literature documents numerous ways that altered proteoglycan metabolism can influence prostate cancer development and progression, including altering prostate cancer cell growth, motility, survival, local diffusion of growth factors, and cell signaling [
34]. The enrichment of GAG metabolism, and specifically heparan sulfate and chondroitin sulfate metabolism, in regions with lower DNA methylation in benign-adjacent prostate tissues likely points to the structural changes occurring in the extracellular space surrounding the cancer, and we confirmed that the majority of these genes have higher expression in the benign-adjacent tissues (Additional file
10: Table S5A). A recent study investigating transcriptional activity of genes involved in heparan sulfate biosynthesis in prostate tissues found that these genes have lower expression in prostate cancer tissues compared to prostate tissue from individuals with no prostate cancer, and findings from our study suggest that the expression of these genes is down-regulated in prostate cancer, at least in part, due to epigenetic changes [
35].
Regions of the genome with reduced DNA methylation in the prostate cancer tissue were enriched for a diverse collection of cellular pathways. Olfactory signaling was represented among the enriched pathways. We observed a large number of odorant receptor genes had less methylation in their gene regulatory region in the prostate cancer tissue in comparison with benign-adjacent prostate tissue, and their gene expression levels were mostly higher in the cancer tissues (Additional file
10: Table S5B). A recent study demonstrated that activation of odorant receptors increases cell invasion into collagen gel [
36].
We overlaid ENCODE TF ChIP-seq data with sites of differential methylation and observed that EZH2 was the most highly enriched TF binding in these regions. EZH2 is part of the polycomb repressive complex that is known to regulate chromatin structure during development primarily through repression of expression of a large and diverse set of genes [
37]. EZH2 functions to repress gene expression through methylation of histone H3 at lysine 27 (H3K27 methylation), and EZH2 can also recruit DNA methyltransferases to EZH2-target promoters [
31,
38,
39]. EZH2 expression increases throughout prostate cancer progression and EZH2 expression levels are associated with methylation level in prostate cancer [
11,
40]. Our data suggest that EZH2-directed methylation alterations are critical for the formation and maintenance of prostate cancer, in addition to roles EZH2 plays in castration-resistant prostate cancer.
A practical application of genome wide DNA methylation profiling is the identification of candidate diagnostic biomarkers. We have demonstrated that as few as three CpGs can be used to distinguish benign-adjacent from malignant prostate tissues with high sensitivity (92.6%) and specificity (87.8%). Methylation biomarkers have been identified in prostate cancer previously, including promoter segments of GSTP1, RASSF1, and APC, which are used in commercial tissue-based test to identify patients needing repeat biopsies after an initial negative biopsy [
41]. Clinical validation studies of this commercial methylation-based assay obtained a sensitivity level of 68% and a specificity level of 64% [
42,
43]. We also investigated candidate diagnostic models developed from CpGs that have higher methylation in prostate cancer tissue. Hypermethylated CpGs appear to retained throughout all stages of prostate cancer, likely due to selection pressures, whereas CpGs that become hypomethylated in prostate cancer are less likely to be preserved [
44]. In this context, we tested a 3-CpG model that provided a sensitivity of 90% and a specificity of 82% that again, exceed those reported for DNA methylation markers currently in use. One of the diagnostic the diagnostic CpGs (cg00054525) falls within the regulatory region of the
CYBA gene. Methylation of
CYBA has been previously associated with the progression of melanoma [
45,
46]. However, other genes associated with our diagnostic CpGs, such as
HLA-J and a non-coding RNA, have not yet been associated with cancer, to our knowledge, and thus, introduce new biological aspects to explore. Our model’s diagnostic performance is relatively poor in lung, breast and pancreas adenocarcinomas, suggesting it has some specificity to prostate cancer. This is a characteristic that could hold value in future studies pursuing a non-invasive, peripheral fluids-based assay.
It is important to note that currently available prostate cancer patient cohorts, including our own, are limited in numbers of samples, and future studies will elucidate the value of our DNA methylation signatures across larger cohorts of prostate cancer patients. Furthermore, the full utility of these DNA methylation-based diagnostic biomarkers will be realized when they can be measured in peripheral fluids from patients. Thus, an important future direction of our study is to determine whether these DNA methylation signatures can be detected in patient urine or blood. To definitively determine their clinical relevance, it will be important to directly compare these diagnostic biomarkers to clinically established markers, such as PSA. Finally, given the recent identification of molecular subtypes of prostate cancer, it will be important to determine DNA methylation patterns that not only distinguish tumor tissue from benign tissue, but also can inform about the molecular subtype of the tumor [
17].
Acknowledgements
We thank the HudsonAlpha Genomic Services Lab for providing RNA sequencing data for this project. We thank Dr. Jason Gertz, Dr. Katherine Varley, and Dr. Kevin Bowling for stimulating discussions and critical reading of the manuscript. We thank Joshua Lasseigne for his help in analyzing differential methylation of transcription factor binding sites. We thank Kevin Roberts and Krista Stanton for assistance with the Illumina array. We acknowledge use of The Cancer Genome Atlas project prostate datasets, which were extremely valuable in validation of our findings.