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DNA Methylation for Predicting Prostate Cancer: Where Do We Stand?

By Stuart P. Atkinson, Ph.D.

December 13, 2022

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Introduction

In some instances of prostate cancer, the second-most common cancer diagnosed in men, the disease can rapidly develop into a metastatic and highly lethal form. The identification of novel prognostic biomarkers could enable earlier interventions and the application of more personalized therapeutic approaches, which could support improved outcomes for these men.

Currently employed clinicopathological prognostic factors generally lack sensitivity and specificity in predicting the progression or outcome of prostate cancer. As Susan J. Clark (Garvan Institute of Medical Research, Sydney, New South Wales, Australia), Head of the Epigenetic Research lab and lead researcher of a recent study, notes, "There's a need for men with prostate cancer to have more personalized treatments guided by the nature of their tumors, and they can't get that without new biomarkers that can better predict the risk of developing the lethal form the disease."

So, what biomarkers should we be exploring?

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DNA Methylation Analysis as a Source of Biomarkers

Epigenetic alterations can represent early events in the development of tumorigenesis. These changes include alterations in DNA methylation, which possess enhanced stability compared to, for example, looking at RNA for transcriptomic analyses. But could DNA methylation analyses define potential or predictive biomarkers for prostate cancer patients?

Previously published research had linked DNA methylation status to prostate cancer prognosis (Kristiansen, 2018 and Lam et al., 2020); however, most of these studies had focused on prognostic methylation markers predicting the increasing levels of prostate-specific antigen in the blood of prostate cancer patients after treatment with surgery or radiation ("biochemical recurrence") even given the fact that this measure does not always represent a suitable surrogate for prostate cancer-specific mortality (Zhang et al. 2018).

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Genome-wide Profiling of DNA Methylation in Prostate Cancer Patients - A Source of New Biomarkers

In the hope of developing more reliable and accurate prognostic biomarkers to guide personalized disease management, the research team led by Susan J. Clark evaluated genome-wide patterns of DNA methylation at single-base resolution in prostate cancer patients where there was also long-term clinical follow-up data.

Specifically, the authors employed whole-genome bisulfite sequencing to compare DNA methylation profiles in surgically removed primary prostate cancer tissues (radical prostatectomy) in patients with both lethal and non-lethal forms of the disease that and also possessed median follow-up data of nearly twenty years. Subsequently, they employed targeted multiplex bisulfite sequencing of candidate regions to validate any detected differentially methylated regions in an independent cohort with median follow-up data of fifteen years. Of importance, long-term follow-up times represent clinically important endpoints, as they allow sufficient time for metastatic relapse and prostate cancer-specific mortality to manifest (Albertsen et al. 2005).

The whole-genome bisulfite sequencing data analysis identified various cancer-specific DNA methylation patterns (including CpG island hypermethylation and hypomethylation of repetitive elements, which are well understood as cancer-associated hallmarks) that are associated with increased disease risk. Initial principal component analysis of the vast amounts of data not only demonstrated that significant differences separated normal and tumor samples, but also tumors from patients with lethal and non-lethal forms of the disease. The authors then identified a set of differentially methylated regions between patients who died less than ten years after surgery and those still alive more than ten years after surgery. In summary, over 1400 differentially methylated regions associated with prostate cancer-specific mortality, with most of these loci located in promoter and CpG island regions (generally regions that regulate gene expression) associated with genes known to be downregulated in prostate cancer (Liu et al. 2006). Most differentially methylated regions displayed hypermethylation, suggesting that the identified prostate cancer development-associated alterations prompted gene silencing.

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Validating DNA Methylation Biomarkers for Prostate Cancer

The team then selected and validated top-ranked, differentially methylated regions associated with disease risk in a large, independent clinical cohort (Zhang et al. 2018 and Grogan et al. 2017). These regions included the CpG island-containing promoters of genes previously associated with prostate cancer progression, such as calcium release activated channel regulator 2a (CRACR2A) (Dai et al. 2019), cysteine dioxygenase type 1 (CDO1) (Meller et al. 2016), and T-box transcription factor 1 (TBX1) (Lin et al. 2013 and Al Olama et al. 2014). Additional validation of the prognostic utility of the top-ranked eighteen differentially methylated regions associated with disease risk employed targeted multiplex bisulfite PCR sequencing in an independent cohort to evaluate associations with biochemical recurrence, metastatic recurrence, and prostate cancer-specific mortality.

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CACNA2D4 Methylation: A Novel and Crucial Biomarker?

Of note, DNA methylation at a locus associated with the calcium channel, voltage-dependent, alpha 2/delta subunit 4 (CACNA2D4) gene displayed a significant association with all three endpoints; therefore, differential methylation at this region was selected for further analysis in multivariable models for biochemical recurrence and prostate cancer-specific mortality as a significant independent prognostic variable. This approach improved the prediction of prostate cancer-specific mortality in a manner comparable with improvements afforded by the inclusion of data from commercially available gene expression prognostic biomarker tests. The authors described an overlap of the CACNA2D4 differentially methylated region with a regulatory element known to target the CACNA2D4 promoter, suggesting that methylation may play a role in long-range transcriptional regulation of the CACNA2D4 gene. However, they also note that a more in-depth analysis of this region's regulatory mechanisms would require further study through techniques like Hi-C to capture chromatin conformation and CRISPR interference.

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Improving Prostate Cancer Management with the Help of Epigenetic Biomarkers

These data describe the DNA methylation profiles associated with non-lethal and lethal forms of prostate cancer and have helped to define distinguishing genic regions; excitingly, the authors also report that including these DNA methylation biomarkers can improve prognostic models of prostate cancer mortality and, therefore, hold promise for clinical application. Ongoing research must next validate the novel panel of epigenetic biomarkers described (including the predictive value of the CACNA2D4 locus) and determine if they describe the disruption of critical regulatory pathways that support prostate cancer metastases and death.

Lisa Horvath, fellow Garvan Institute researcher, oncologist, and clinical lead on the study, concludes, "What you really want to know on the day a patient is diagnosed, is who has the potential for lethal prostate cancer and who doesn't, because it will change the way you treat the cancer. These epigenetic biomarkers have the potential to help us work out up front who has lethal prostate cancer and who doesn't."

Lisa Horvath, fellow Garvan Institute researcher, oncologist, and clinical lead on the study, concludes, "What you really want to know on the day a patient is diagnosed, is who has the potential for lethal prostate cancer and who doesn't, because it will change the way you treat the cancer. These epigenetic biomarkers have the potential to help us work out up front who has lethal prostate cancer and who doesn't."

For more details on this exciting epigenetic study into the predictive power of DNA methylation, see Clinical and Translational Medicine, September 2022.

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About the author

Stuart P. Atkinson, Ph.D.

Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!

Contact Stuart on Twitter with any questions

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