Identification of key pathways and genes with aberrant methylation in prostate cancer using bioinformatics analysis

Figures & data

Table 1 Differentially methylated genes (DMGs) shortlisted for analysis

S no	Shortlisted DMGs associated with PCa
1	Adenomatous polyposis coli (APC)
2	Caveolin 1 (CAV1)
3	Cluster of differentiation 44 (CD44)
4	Cadherin 13 (CDH13)
5	Cyclin-dependent kinase inhibitor 1C (CDKN1C)
6	Death-associated protein kinase 1 (DAPK1)
7	Endothelin receptor type B (EDNRB)
8	Estrogen receptor 2 (ESR2)
9	Glutathione peroxidase 3 (GPX3)
10	Hypermethylated in cancer 1 (HIC1)
11	Prostaglandin-endoperoxide synthase 2 (PTGS2)
12	Ras association domain family member 1 (RASSF1)
13	Stratifin (SFN)
14	Androgen receptor (AR)
15	Cyclin D2 (CCND2)
16	Cadherin 1 (CDH1)
17	Cyclin-dependent kinase inhibitor 1B (CDKN1B)
18	Cyclin-dependent kinase inhibitor 2A (CDKN2A)
19	Dickkopf WNT signaling pathway inhibitor (DKK3)
20	Estrogen receptor 1 (ESR1)
21	Fragile histidine triad (FHIT)
22	Glutathione S-transferase pi 1 (GSTP1)
23	O-6-methylguanine-DNA methyltransferase (MGMT)
24	Retinoic acid receptor beta (RARβ)
25	S100 calcium-binding protein A6 (S100A6)
26	TIMP metallopeptidase inhibitor 3 (TIMP3)

Table 2 Gene ontology (GO) analysis of candidate differentially methylated genes (DMGs) associated with prostate cancer (PCa)

Annotation	Name	Gene count	p-value	Genes involved
Molecular functions (MFs)
GO:0005515	Protein binding	16	1.0633E–06	S100A6, TIMP3, CD44, CDKN1C, CAV1, ESR1, CDKN1B, RASSF1, CDH1, SFN, ESR2, AR, DAPK1, FHIT, GSTP1, HIC1
GO:0008270	Zinc ion binding	7	0.0042104	S100A6, ESR1, RASSF1, ESR2, AR, HIC1, RARβ
GO:0046872	Metal ion binding	8	0.00454845	TIMP3, ESR1, RASSF1, ESR2, PTGS2, AR, HIC1, RARβ
GO:0004872	Receptor activity	6	0.00461516	CD44, EDNRB, ESR1, ESR2, AR, RARβ
GO:0003677	DNA binding	6	0.00644885	ESR1, ESR2, CD44, EDNRB, AR, RARβ
Cellular localization
GO:0005737	Cytoplasm	20	4.1853E–10	S100A6, TIMP3, CD44, CDKN1C, CAV1, ESR1, CDKN1B, RASSF1, CDH13, CDH1, SFN, CDKN2A, APC, PTGS2, AR, DAPK, FHIT, CCND2, GSTP1, RARβ
GO:0005634	Nucleus	16	6.62952E–06	S100A6, CDKN1C, ESR1, CDKN1B, RASSF1, SFN, ESR2, MGMT, APC, PTGS2, AR, FHIT, CCND2, GSTP1, HIC1, RARβ
GO:0005654	Nucleoplasm	7	8.58777E–05	ESR1, CDKN1B, ESR2, CDKN2A, MGMT, AR, RARβ
GO:0005886	Plasma membrane	10	0.00182238	S100A6, CD44, EDNRB, ESR1, RASSF1, CDH13, CDH1, FHIT, GSTP1, CAV1
GO:0005829	Cytosol	6	0.019808	S100A6, CAV1, CDKN1B, FHIT, CCND2, GSTP1
Biological process (BP)
GO:0042493	Response to drugs	6	1.86731E–05	GPX3, CAV1, CDH1, MGMT, APC, PTGS2
GO:0008285	Negative regulation of cell proliferation	6	1.82718E–07	CDKN1C, CAV1, CDKN1B, CDH13, SFN, PTGS2
GO:0043066	Negative regulation of apoptotic process	5	0.000106969	CD44, CDKN1B, APC, AR, GSTP1
GO:0007050	Cell cycle arrest	4	0.000128057	CDKN1C, CDKN1B, RASSF1, CDKN2A
GO:0032355	Response to estradiol stimulus	4	5.21837E–05	ESR1, PTGS2, CCND2, GSTP1
GO:0014070	Response to organic cyclic compound	4	0.000109315	TIMP3, GPX3, PTGS2, CCND2
GO:0007165	Signal transduction	7	0.00051767	S100A6, ESR1, SFN, ESR2, AR, DAPK1, RARβ
KEGG-enriched pathways
(KEGG)05200	Pathways in cancer	10	1.00997E–12	RARβ, AR, CDKN1B, APC, GSTP1, RASSF1, DAPK1, CDKN2A, CDH1, PTGS2
(KEGG)04110	Cell cycle	5	4.91434E–07	CCND2, CDKN1B, SFN, CDKN2A, CDKN1C
(KEGG)04115	p53 signaling	3	0.000129557	CCND2, SFN, CDKN2A
(KEGG)05215	PCa	3	0.000251702	AR, CDKN1B, GSTP1
(KEGG)04310	Wnt signaling	2	0.0189158	CCND2, APC
(KEGG)04510	Focal adhesion	2	0.0300763	CCND2, CAV1

Figure 1 Protein–protein interaction (PPI) network of identified differentially methylated genes (DMGs).

Table 3 Associated genes of identified seven differentially methylated hub genes

Hub gene	Connected genes
AR	ESR1, CAV1, ESR2, CDH1, RARβ, CDKN1B
CDH13	CDH1, RASSF1, DAPK1, GSTP1, CDKN2A, RARβ, FHIT, MGMT, TIMP3, HIC1
CDKN2A	CCND2, CDKN1B, RASSF1, FHIT, CDH1, MGMT, GSTP1, ESR1, TIMP3, HIC1, RARβ, SFN, AR, ESR2
DAPK1	RASSF1, TIMP3, RARβ, MGMT, HIC1, FHIT, CDH1
GSTP1	GPX3, TIMP3, RASSF1, DAPK1, MGMT, CDH1, RARβ, HIC1, ESR1, FHIT, SFN, PTGS2, AR, CCND2, ESR2
RASSF1	FHIT, SFN, RARβ, MGMT, HIC1, TIMP3, CDH1, CCND2, ESR1, ESR2
CD44	AR, CDKN1B, CAV1, ESR1, CDKN2A, TIMP3, PTGS2

Table 4 Methylation and Expression database of Normal and Tumor tissues (MENT) data showing correlation between mean methylation values and gene expressions

Gene name	Methylation	Probe	Correlation
CDH13	ILMN_1766925	cg 13759328	−0.53224
DAPK1	ILMN_1708340	cg 08797471	−0.75606
RASSF1	ILMN_1808066	cg 06821120	−0.44059
AR	ILMN_1792540	cg 05786601	−0.56984
CDKN2A	ILMN_1717714	cg 11653709	−0.68581
GSTP1	ILMN_1679869	cg 05244766	−0.55374
CD44	ILMN_1778625	cg 04125208	−0.39674

Table 5 Specificity and sensitivity of hub genes identified in our study that are frequently hypermethylated as potential biomarkers for prostate cancer (PCa)

S no	Gene name	Sample type	Sensitivity	Specificity	Significance/stage	References
1	GSTP1	Biopsy, ejaculate, serum, urine, urine post-massage	27%–100%	80%–100%	All stages	^Citation22^–^Citation34
2	DAPK	Urine post-massage	93%	Not determined	Primary and metastatic	^Citation35^–^Citation38
3	RASSF1	Serum, urine, urine post-massage	80%–100%	89%–100%	Prostatic intraepithelial neoplasia (PIN), primary and metastatic	^Citation35
4	CDH13	Tissue	72.30%	48%	Recurrence	^Citation36
5	CDKN2A	Urine	87%	100%	PIN, primary and metastatic	^Citation34, ^Citation39
6	CD44	Tissues	87%	Not determined	All stages, recurrence	^Citation40, ^Citation41
7	AR	Urine, tissue	8%–28%	Not determined	All stages	^Citation42^–^Citation45

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