Multicenter Study

. 2020 Aug;52(8):778-789.

doi: 10.1038/s41588-020-0648-8. Epub 2020 Jul 13.

The DNA methylation landscape of advanced prostate cancer

Shuang G Zhao^#^{1

2

3

4

5}, William S Chen^#^{3

4

6}, Haolong Li^#^{3

4}, Adam Foye^{4

7}, Meng Zhang^{3

4}, Martin Sjöström^{3

4}, Rahul Aggarwal^{4

7}, Denise Playdle^{4

7}, Arnold Liao⁸, Joshi J Alumkal^{9

10}, Rajdeep Das^{3

4}, Jonathan Chou^{3

4

7}, Junjie T Hua¹¹, Travis J Barnard^{3

4}, Adina M Bailey^{4

7}, Eric D Chow^{12

13}, Marc D Perry^{3

4

7}, Ha X Dang^{14

15

16}, Rendong Yang¹⁷, Ruhollah Moussavi-Baygi^{3

4}, Li Zhang^{4

7}, Mohammed Alshalalfa³, S Laura Chang³, Kathleen E Houlahan^{18

19

20}, Yu-Jia Shiah¹⁸, Tomasz M Beer^{9

21}, George Thomas^{9

22}, Kim N Chi^{23

24}, Martin Gleave²³, Amina Zoubeidi²³, Robert E Reiter²⁵, Matthew B Rettig^{25

26}, Owen Witte²⁷, M Yvonne Kim²⁸, Lawrence Fong^{4

7}, Daniel E Spratt¹, Todd M Morgan^{2

5

29}, Rohit Bose^{4

7

30

31}, Franklin W Huang^{4

7}, Hui Li^{3

4}, Lisa Chesner^{3

4}, Tanushree Shenoy^{4

7}, Hani Goodarzi^{12

30}, Irfan A Asangani³², Shahneen Sandhu³³, Joshua M Lang³⁴, Nupam P Mahajan^{16

35}, Primo N Lara^{36

37}, Christopher P Evans^{37

38}, Phillip Febbo⁸, Serafim Batzoglou⁸, Karen E Knudsen³⁹, Housheng H He^{11

19}, Jiaoti Huang⁴⁰, Wilbert Zwart⁴¹, Joseph F Costello²⁸, Jianhua Luo⁴², Scott A Tomlins⁵, Alexander W Wyatt²³, Scott M Dehm^{43

44}, Alan Ashworth^{4

7}, Luke A Gilbert^{4

30}, Paul C Boutros^{19

20

25}, Kyle Farh⁸, Arul M Chinnaiyan^{2

5

29

45

46

47}, Christopher A Maher^{14

15

16

48}, Eric J Small^{4

7}, David A Quigley^{4

30

49}, Felix Y Feng^{50

51

52

53}

Affiliations

¹ Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA.
² Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA.
³ Department of Radiation Oncology, University of California San Francisco, San Francisco, CA, USA.
⁴ Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA.
⁵ Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, USA.
⁶ Yale School of Medicine, New Haven, CT, USA.
⁷ Division of Hematology and Oncology, Department of Medicine, University of California San Francisco, San Francisco, CA, USA.
⁸ Illumina Inc., San Diego, CA, USA.
⁹ Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA.
¹⁰ Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA.
¹¹ Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
¹² Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA.
¹³ Center for Advanced Technology, University of California San Francisco, San Francisco, CA, USA.
¹⁴ McDonnell Genome Institute, Washington University, St. Louis, MO, USA.
¹⁵ Department of Internal Medicine, Washington University, St. Louis, MO, USA.
¹⁶ Siteman Cancer Center, Washington University, St. Louis, MO, USA.
¹⁷ The Hormel Institute, University of Minnesota, Austin, MN, USA.
¹⁸ Ontario Institute for Cancer Research, Toronto, Ontario, Canada.
¹⁹ Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
²⁰ Department of Human Genetics, Institute for Precision Health, UCLA, Los Angeles, CA, USA.
²¹ Division of Hematology/Medical Oncology, Department of Medicine, Oregon Health & Science University, Portland, OR, USA.
²² Department of Pathology, Oregon Health & Science University, Portland, OR, USA.
²³ Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
²⁴ British Columbia Cancer Agency, Vancouver Centre, Vancouver, British Columbia, Canada.
²⁵ Jonsson Comprehensive Cancer Center, Departments of Medicine and Urology, University of California Los Angeles, Los Angeles, CA, USA.
²⁶ Department of Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA.
²⁷ Department of Microbiology, Immunology, and Molecular Genetics at the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
²⁸ Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, USA.
²⁹ Department of Urology, University of Michigan, Ann Arbor, MI, USA.
³⁰ Department of Urology, University of California San Francisco, San Francisco, CA, USA.
³¹ Department of Anatomy, University of California San Francisco, San Francisco, CA, USA.
³² Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA.
³³ Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia.
³⁴ Department of Medicine, University of Wisconsin, Madison, WI, USA.
³⁵ Department of Surgery, Washington University, St. Louis, MO, USA.
³⁶ Division of Hematology Oncology, Department of Internal Medicine, University of California Davis, Sacramento, CA, USA.
³⁷ Comprehensive Cancer Center, University of California Davis, Sacramento, CA, USA.
³⁸ Department of Urologic Surgery, University of California Davis, Sacramento, CA, USA.
³⁹ Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA.
⁴⁰ Department of Pathology, Duke University, Durham, NC, USA.
⁴¹ Netherlands Cancer Institute, Oncode Institute, Amsterdam, the Netherlands.
⁴² Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA.
⁴³ Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
⁴⁴ Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA.
⁴⁵ Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
⁴⁶ Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA.
⁴⁷ Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, MI, USA.
⁴⁸ Department of Biomedical Engineering, Washington University, St. Louis, MO, USA.
⁴⁹ Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA, USA.
⁵⁰ Department of Radiation Oncology, University of California San Francisco, San Francisco, CA, USA. Felix.Feng@ucsf.edu.
⁵¹ Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA. Felix.Feng@ucsf.edu.
⁵² Division of Hematology and Oncology, Department of Medicine, University of California San Francisco, San Francisco, CA, USA. Felix.Feng@ucsf.edu.
⁵³ Department of Urology, University of California San Francisco, San Francisco, CA, USA. Felix.Feng@ucsf.edu.

^# Contributed equally.

PMID: 32661416
PMCID: PMC7454228
DOI: 10.1038/s41588-020-0648-8

Multicenter Study

The DNA methylation landscape of advanced prostate cancer

Shuang G Zhao et al. Nat Genet. 2020 Aug.

. 2020 Aug;52(8):778-789.

doi: 10.1038/s41588-020-0648-8. Epub 2020 Jul 13.

Authors

Affiliations

¹ Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA.
² Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA.
³ Department of Radiation Oncology, University of California San Francisco, San Francisco, CA, USA.
⁴ Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA.
⁵ Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, USA.
⁶ Yale School of Medicine, New Haven, CT, USA.
⁷ Division of Hematology and Oncology, Department of Medicine, University of California San Francisco, San Francisco, CA, USA.
⁸ Illumina Inc., San Diego, CA, USA.
⁹ Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA.
¹⁰ Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA.
¹¹ Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
¹² Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA.
¹³ Center for Advanced Technology, University of California San Francisco, San Francisco, CA, USA.
¹⁴ McDonnell Genome Institute, Washington University, St. Louis, MO, USA.
¹⁵ Department of Internal Medicine, Washington University, St. Louis, MO, USA.
¹⁶ Siteman Cancer Center, Washington University, St. Louis, MO, USA.
¹⁷ The Hormel Institute, University of Minnesota, Austin, MN, USA.
¹⁸ Ontario Institute for Cancer Research, Toronto, Ontario, Canada.
¹⁹ Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
²⁰ Department of Human Genetics, Institute for Precision Health, UCLA, Los Angeles, CA, USA.
²¹ Division of Hematology/Medical Oncology, Department of Medicine, Oregon Health & Science University, Portland, OR, USA.
²² Department of Pathology, Oregon Health & Science University, Portland, OR, USA.
²³ Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
²⁴ British Columbia Cancer Agency, Vancouver Centre, Vancouver, British Columbia, Canada.
²⁵ Jonsson Comprehensive Cancer Center, Departments of Medicine and Urology, University of California Los Angeles, Los Angeles, CA, USA.
²⁶ Department of Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA.
²⁷ Department of Microbiology, Immunology, and Molecular Genetics at the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
²⁸ Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, USA.
²⁹ Department of Urology, University of Michigan, Ann Arbor, MI, USA.
³⁰ Department of Urology, University of California San Francisco, San Francisco, CA, USA.
³¹ Department of Anatomy, University of California San Francisco, San Francisco, CA, USA.
³² Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA.
³³ Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia.
³⁴ Department of Medicine, University of Wisconsin, Madison, WI, USA.
³⁵ Department of Surgery, Washington University, St. Louis, MO, USA.
³⁶ Division of Hematology Oncology, Department of Internal Medicine, University of California Davis, Sacramento, CA, USA.
³⁷ Comprehensive Cancer Center, University of California Davis, Sacramento, CA, USA.
³⁸ Department of Urologic Surgery, University of California Davis, Sacramento, CA, USA.
³⁹ Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA.
⁴⁰ Department of Pathology, Duke University, Durham, NC, USA.
⁴¹ Netherlands Cancer Institute, Oncode Institute, Amsterdam, the Netherlands.
⁴² Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA.
⁴³ Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
⁴⁴ Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA.
⁴⁵ Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
⁴⁶ Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA.
⁴⁷ Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, MI, USA.
⁴⁸ Department of Biomedical Engineering, Washington University, St. Louis, MO, USA.
⁴⁹ Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA, USA.
⁵⁰ Department of Radiation Oncology, University of California San Francisco, San Francisco, CA, USA. Felix.Feng@ucsf.edu.
⁵¹ Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA. Felix.Feng@ucsf.edu.
⁵² Division of Hematology and Oncology, Department of Medicine, University of California San Francisco, San Francisco, CA, USA. Felix.Feng@ucsf.edu.
⁵³ Department of Urology, University of California San Francisco, San Francisco, CA, USA. Felix.Feng@ucsf.edu.

^# Contributed equally.

PMID: 32661416
PMCID: PMC7454228
DOI: 10.1038/s41588-020-0648-8

Abstract

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.

PubMed Disclaimer

Figures

**Figure 1:. CpG Methylator Phenotype (CMP)**
a, Sample-level summary of hypo-methylated region (HMR) frequency and somatic alterations in 100 independent mCRPC samples. Bar plots show HMR counts within genomic features (HMR count), counts of HMRs overlapping with CpG islands/shores/shelves (CpG overlap), percent of the genome with DNA copy number alterations (CNA %), somatic mutations per megabase (Mutations / Mb), and counts of structural variants (SV count). CMP samples labeled in blue. TFBS, transcription factor binding site. b, Hierarchical clustering of the 10% most variable recurrent HMRs in 100 mCRPC samples. Blue dendrogram denotes CMP samples. c, HMR count per sample in thousands in non-CMP (N=78) and CMP (N=22). Significance was assessed with two-sided Wilcoxon test. d, Percent of CpGs methylated at loci harboring recurrent HMRs in Non-CMP (N=78) and CMP (N=22), plotted and assessed as in (c). e, rHMRs located in CpG islands, shores, and shelves, count per sample in thousands, plotted and assessed as in (c). f, rHMRs located in open seas, count per sample in thousands, plotted and assessed as in (c). Boxplots show the median, first, and third quartiles, and outliers are shown if outside 1.5x the inter-quartile range.

**Figure 2:. DNA methylation valleys (DMVs)**
a, Top: Sample-level log₂ odds ratio calculated from the number of DMVs which overlap H3K4me3 vs. H3K27me3 sites. Lower values favor H3K27me3, higher values favor H3K4me3. Bottom: Sample-level count of DMVs in order matching top panel. b, Mean percent methylation across the AR locus for benign prostate (N=4), localized prostate cancer (N=5), mCRPC adenocarcinoma (N=95), and t-SCNC samples (N=5). Vertical black lines show the location of the previously identified AR enhancer. The vertical green and red lines show the TSS and transcriptional terminator of the androgen receptor, respectively.

**Figure 3:. Methylation associated with prostate cancer-specific genes**
a, Variability in gene expression levels versus the correlation between gene expression and methylation. Expression variability was calculated as standard deviation (Log₂(TPM+1), and correlation calculated at the most significant promoter/gene body eHMR for each gene. Y-axis box-plot shows gene expression variability for prostate cancer-specific genes versus all other genes. X-axis box-plot shows correlation of methylation with gene expression of prostate cancer-specific genes versus all other genes. Significance was assessed with two-sided Wilcoxon test, N=169 vs. 51502. Boxplots show the median, first, and third quartiles, and outliers are shown if outside 1.5x the inter-quartile range. b, Sample-level gene expression levels compared to the presence of DNA alterations and methylation at the most significant promoter/gene body eHMR. Alterations predicted to be activating (*SLC45A3*, *SPON2*, *TDRD1*, *SCHLAP1*) or inactivating (*TMEFF2*, *PCAT14*) are shown). Significance of methylation levels was assessed by ANOVA comparing a model predicting gene expression from DNA alterations alone to a second model with methylation as an added factor. N=100 independent mCRPC samples. CN, copy number.

**Figure 4:. Methylation association with the androgen response pathway**
a, Percentage of genes in MSigDB Hallmark pathways for which methylation predicted expression independently from DNA alterations in a linear model. An asterisk indicates significant enrichment (two-sided FDR ≤ 0.05) relative to the set of all housekeeping genes. Significance was assessed with a two-sided Fisher’s exact test. N=100 independent mCRPC samples. b, Sample-level gene expression levels compared to the presence of DNA alterations and methylation at the most significant promoter/gene body eHMR. Alterations predicted to be activating (*KLK3*, *FOLH1*) or inactivating (*NKX3–1*) are shown). Significance of methylation levels was assessed by ANOVA comparing a model predicting gene expression from DNA alterations alone to a second model with methylation as an added factor, N=100 independent mCRPC samples. c, HMRs, correlation between methylation in at loci harboring recurrent HMRs and AR expression, ChIP-seq peaks (H3K27ac, AR, ERG, FOXA1, HOXB13), and ChIA-PET interactions (AR and ERG) at the AR locus. Stars denote HMRs at which methylation was associated with AR expression (eHMRs), colored black for previously reported AR upstream enhancer, blue for the AR promoter, gold for new putative AR regulatory regions. Significance was assessed with a two-sided Spearman’s correlation test, N=100 independent mCRPC samples. “Primary” in the ChIP-seq tracks indicates localized primary prostate cancer.

Figure 5:. Methylation association with *TMPRSS2*-*ERG* and *MYC*
a, HMRs, correlation between methylation in loci harboring recurrent HMRs and *ERG* expression, and ChIP-seq peaks (H3K27ac, AR, ERG, FOXA1, HOXB13) at the *TMPRSS2* locus. Significance was assessed with two-sided Spearman’s correlation, N=100 independent mCRPC samples. *TMPRSS2* isoform 204 was not shown as its TSS was ~20Kbp upstream of the other 5 protein coding isoforms. b, Observed *ERG* expression in *TMPRSS2-ERG* fusion positive mCRPC and *ERG* expression predicted in those tumors using two linear models: one including AR expression and AR mutations and another including AR expression, AR mutations, and methylation at the *TMPRSS2* promoter and upstream locus. Significance was assessed by a two-sided ANOVA (N=41 independent fusion positive samples). c, HMRs, correlation between methylation in recurrent HMRs and *MYC* expression, ChIP-seq peaks (H3K27ac), and ChIA-PET interactions (AR and ERG) at the *MYC*-*PVT1* locus. Significance was assessed with two-sided Spearman’s correlation, N=100 independent mCRPC samples. “Primary” in the ChIP-seq tracks indicates localized primary prostate cancer. d, Observed *MYC* expression and *MYC* expression predicted in those tumors using two linear models: one including *MYC* copy number alone and another including *MYC* copy number and methylation at the *MYC*-*PVT1* locus. Significance was assessed by a two-sided ANOVA (N=100 independent mCRPC samples).

**Figure 6:. Genome-wide analysis of differential methylation**
a, Differentially methylated regions (DMRs) and mutation frequency in mCRPC. Ideogram shows, for each chromosome, from left to right: DMRs comparing primary prostate cancer (N=5) to benign prostate (N=4), DMRs comparing mCRPC (adenocarcinoma, N=95) to primary prostate cancer (N=5), and mutational frequency in 1Mbp windows in the mCRPC samples (excluding two hyper-mutated samples). Maximum bar height in mutation frequency represents an average mutational frequency ≥10 mutations per Mb per sample. b, Differential methylation (comparing mCRPC (adenocarcinoma) to benign prostate) compared to mutational frequency (excluding 2 hyper-mutated samples), N=98. Each point represents a fixed 1Mbp window of the genome, and all points collectively represent all 1 Mb windows across the genome excluding centromeres and telomeres. c, Average differential methylation values across all sites identified from publicly available ChIP-seq data (AR, *ERG*, *FOXA1*, *HOXB13*, H3K27ac). For each ChIP-seq peak, a 20Kbp window centered on midpoint of the peak (x=0) was assessed for differential methylation between mCRPC adenocarcinoma vs. benign prostate samples.

See this image and copyright information in PMC

References

1. Jones PA & Baylin SB The fundamental role of epigenetic events in cancer. Nat Rev Genet 3, 415–28 (2002). - PubMed
1. Feinberg AP, Koldobskiy MA & Gondor A Epigenetic modulators, modifiers and mediators in cancer aetiology and progression. Nat Rev Genet 17, 284–99 (2016). - PMC - PubMed
1. Okano M, Bell DW, Haber DA & Li E DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–57 (1999). - PubMed
1. Rechache NS et al. DNA methylation profiling identifies global methylation differences and markers of adrenocortical tumors. J Clin Endocrinol Metab 97, E1004–13 (2012). - PMC - PubMed
1. Skvortsova K et al. DNA Hypermethylation Encroachment at CpG Island Borders in Cancer Is Predisposed by H3K4 Monomethylation Patterns. Cancer Cell 35, 297–314 e8 (2019). - PubMed

Publication types

Actions
Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- The Lens - Patent Citations Database
Medical
- MedlinePlus Health Information
Research Materials
- NCI CPTC Antibody Characterization Program
Miscellaneous
- NCI CPTAC Assay Portal

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

The DNA methylation landscape of advanced prostate cancer

Affiliations

The DNA methylation landscape of advanced prostate cancer

Authors

Affiliations

Abstract

Figures

References

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Research Materials

Miscellaneous