Table 3.

Associations between NR3C1/HSD11β2 methylation and cancers.

Authors	Study design	Gene	Method	Tissue type	Exposure/Outcome	Sample	Results
Nesset et al. (2014)	Case-control; in vitro cell lines	NR3C1	Methylation-specific PCR	Breast tissue	None/breast cancer	N = 59 breast cancer patients from Ontario, Canada Cases: tumor tissue samples. Controls: matched normal breast clinical tissue	•Exon 1B is the predominant NR3C1 first exon expressed in normal and cancerous breast tissue •8 (15%) breast cancer tumors showed elevated methylation. The 1B promoter was methylated in 7/8 of these. •No methylation was observed in normal breast tissue •No difference in NR3C1 expression between tumors methylated and unmethylated at the NR3C1 proximal promoter as an aggregate •However, tumors methylated at the 1B promoter showed a 4.6-fold decrease in NR3C1 expression compared with tumors unmethylated at this region
Lien et al. (2006)	Cross-sectional	NR3C1	Methylation-specific PCR	Breast tissue	None/breast cancer	N = 118 GR-immunonegative carcinoma specimens (106 IDCs, 8 ILCs, 1 mucinous carcinoma, 3 DCIS) taken from human breast samples in Taipei City, Taiwan	•NR3C1 was strongly expressed in metaplastic carcinomas (94.4%), but was not expressed in the majority of non-metaplastic carcinomas (98.2%) •NR3C1 exon 1C was uniformly unmethylated in non-metaplastic carcinomas
Kay et al. (2011)	Case-control (cell line and tissue); prospective	NR3C1	Bisulfate sequencing	SCLC cell lines	Small cell lung cancer (SCLC)	Cases: 14 Small Cell Lung Cancer (SCLC) cell lines: Cor L24, Cor L27, Cor L31, Cor L32, Cor L42, Cor L47, Cor L51, Cor L88, Cor L99, Cor L103), DMS 79, DMS 153, HC12, and H 148 Cell line controls: human lung epithelial carcinoma cells A549, human embryonic kidney cells HEK-293, human cervical carcinoma cells HeLa, and human osteosarcoma cells U20S (European Collection of Cell Cultures, Wiltshire, UK). Non-small cell lung cancer (NSCLC) lines NCI-H358 and -H727 (European Collection of Cell Cultures, Wiltshire, UK) and NCI-H23, -H441, -H1299 (American Type Culture Collection, USA) Peripheral blood controls: human peripheral blood mononuclear cells (PBMC) from health donors	•There was a significant difference in methylation levels between SCLC cases and controls at several CpG sites in the 1C promoter (CpG 69 individually, CpG 68 and 69 in combination) as well as a significant difference in methylation levels across the whole 1C promoter region (CpGs1-69) and for all 1C CpGs excluding 68 and 69 •There was a significant association between number of methylated CpGs and NR3C1 protein expression within the panel of 14 SCLC cell lines •After 72 h of treatment with 5′Azadeoxycytidine, NR3C1 mRNA levels increased dramatically in SCLC cells while there was no change in expression in HEK and A549 control cells, and a decrease in NR3C1 in U2OS control cells •While 3/4 SCLC cell lines showed augmented NR3C1 expression after 5′Azadeoxycytidine treatment, only 1/4 NSCLC cell lines showed increased NR3C1 expression
Ahlquist et al. (2008)	Case-control	11 genes, including NR3C1	Quantitative methylation-specific PCR	Colorectal cancer (CRC) and normal colon mucosa tissue	None/colorectal cancer	Cases: 63 adenomas from 52 Norwegian individuals (median age: 67), and 52 carcinomas from 51 patients in 7 hospitals in Oslo, Norway (median age: 70 years) Controls: 21 normal colon mucosa samples (N1) from 20 deceased, cancer-free individuals (median age: 52.5 years), and 18 normal colon mucosa samples (N2) from 18 CRC patients in 7 hospitals in Oslo, Norway (median age: 70.5 years)	•NR3C1 showed an increasing methylation frequency from adenomas to carcinomas •NR3C1 methylation was significantly higher among microsatellite instable (MSI) than among microsatellite stable (MSS) carcinomas (P = 0.001) •Vimentin, ADAMTS1, and MAL are likely to be more suitable biomarkers for early detection because they are unmethylated in normal mucosa from healthy individuals and frequently methylated in carcinomas
Lind et al. (2006)	Cross-sectional (cell lines); case-control (tissue)	Multiple genes, including NR3C1	Methylation-specific PCR, bisulfate sequencing	Cell lines, CRC tissue, and normal colon mucosa tissue	Colon cancer	Cell lines: 20 colon cancer cell lines were included: 9 from microsatellite instable (MSI) tumors (Co115, HCT15, HCT116, LoVo, LS174T, RKO, SW48, TC7, and TC71); 11 from microsatellite stable (MSS) tumors (ALA, Colo320, EB, FRI, HT29, IS1, IS2, IS3, LS1034, SW480, and V9P). 10 cell lines from prostate tissue (N = 3), testicular germ cell tumors (N = 3), and ovary tissues (n = 4) were included, as well as a clinical series of prostate (N = 20) and kidney (N = 20) carcinomas from the University Hospital of Porto, and a series of testicular germ cell tumors (N = 42) from patients admitted to the Norwegian Radium Hospital, Oslo Tissue sample cases: DNA from patients in Norwegian hospitals: 53 colorectal carcinomas (25 MSS and 28 MSI) from 52 patients (mean age 68 years), 63 adenomas (61 MSS and 2 MSI) from 52 patients (mean age 67 years) Tissue sample controls: Normal mucosa samples from 22 colorectal cancer patients (mean age 64 years) taken from distant sites from the primary carcinoma, and 22 normal colorectal mucosa samples from cancer-free individuals (mean age 54 years, including 8 individuals ≥ 60 years)	Cell line results: •The promoters of ADAMTS1, CRABP1, and NR3C1 were hypermethylated in 17/20 (85%), 18/20 (90%), and 7/20 (35%) colon cancer cell lines, respectively. •Promoter hypermethylation of NR3C1 was also significantly associated with reduced gene expression in cancer cell lines. •NR3C1 was unmethylated in subsets of cancer cell lines from prostate, testis, and kidney tissues (N = 82). Tissue results: •The primary carcinomas represented in the nine pairs of matched normal and cancer tissue were methylated in 4/7 (57%) for NR3C1, whereas the corresponding normal mucosa samples were unmethylated •The methylation frequency of NR3C1 was also significantly higher in MSI carcinomas (12/28; 43%) than in MSS carcinomas (1/23; 4%) •NR3C1 methylation was higher among tumors from females (11/27; 41%) than in those from males (2/24; 8%), and 77% (10/13) of NR3C1 methylated tumors were located in the right side of the colon
Wu et al. (2007)	Cross-sectional (cell line and tissue)	13 genes, including NR3C1	Methylation-specific PCR, bisulfate sequencing	Ovarian cancer cell lines and ovarian tumor tissue	Ovarian cancer	Cell lines: 4 ovarian carcinoma cell lines, ES-2, OV-90, OVCAR-3, and SKOV-3 (American Type Culture Collection, Manassas, USA) Tissue sample: primary ovarian carcinomas from women (n = 52; mean age: 58 years) displaying a similar distribution regarding FIGO stage and histology. Taken from a tissue bank in Oslo, Norway. Included 19 serous, 5 mucous, 5 clear cell, 17 endometrioid, and 6 of mixed histotype tumors. 2 benign and 2 borderline ovarian tumors were also included	NR3C1 promoter was unmethylated in ovarian carcinoma tissues and in all cell lines
Kang et al. (2008)	Case-control (cell line and tissue)	17 genes, including NR3C1	Quantitative methylation-specific PCR (MethyLight)	Gastric cancer cell lines and normal gastric mucosa tissue	Gastric cancer	Cell line cases: 8 gastric cancer (GC) cell lines treated with 5-aza-deoxycytidine and or Trichostatin A Tissue sample controls: gastric carcinoma-associated non-neoplastic mucosae tissues (GCN) from 33 gastric carcinoma patients in Seoul, Korea (median age: 63 years; male to female ratio 4:1). Normal mucosa tissue from 27 chronic gastritis (CG) patients in Seoul, Korea (median age: 53 years; male to female ratio 19:10). 13 samples were H·Pylori (HP)-negative and 14 samples were HP-positive	•NR3C1 was methylated in GC at a level three times higher than GCN, although this difference was not significant •No significant difference in methylation level in NR3C1 was found between HR-positive and HR-negative CG patients •An inverse relationship between NR3C1methylation and GC cell line RNA expression was established (greater expression seen due to low, non-significant methylation)
Hiraki et al. (2011)	Case-control (cell line and tissue)	Multiple genes, including NR3C1	Quantitative methylation-specific PCR (MethyLight)	Gastric cancer cell lines and normal gastric mucosa tissue	Gastric cancer	Cell lines: 8 gastric cancer cell lines, HSC45, HSC57, KATO III, MKN1, MKN7, MKN28, MKN45, and MKN74 Tissue samples: 20 pairs of adjacent normal gastric mucosa and primary gastric cancer samples. Peritoneal fluid (PF) from 107 patients who underwent surgery in Saga, Japan (median age: 66·5 years; 37 female, 70 male; intestinal type gastric cancer: 46; diffuse type gastric cancer: 61)	•Six genes (BNIP3, CHFR, CYP1B1, MINT25, RASSF2, and SFRP2) showed significantly higher methylation in primary cancer tissues than the adjacent normal gastric mucosa, but methylation of NR3C1 showed no significant difference in methylation levels between the two •NR3C1 methylation was thus deemed not to be cancer-specific
Sanchez-Vega and Gandhi (2009)	Cross-sectional (cell line only)	NR3C1	5-aza-deoxycytidine (decitabine) treatment	Myeloma cell lines	Multiple Myeloma	Myeloma cell line (MM.1) taken from peripheral blood cells of a US patient in the leukemic phase of multiple myeloma (MM) being treated with glucocorticoids (GCs). 1 GC-sensitive cell line, MM.1S, and two GC-resistant cell lines, MM.1RE (early phenotype) and MM.1RL (late phenotype)	•Treating MM.1 cell lines with the demethylating agent 5-aza-deoxycytidine (decitabine) did not change the levels of expression of full length NR3C1 mRNA, thus ruling out the possibility of promoter methylation as a mechanism for decline in NR3C1 mRNA expression in MM.1RL •Other epigenetic observations: histone H3 was trimethylated on Lys4 (3 mK4) in exons 1C, 2, and 3 throughout intron B of NR3C1 RNA in MM.1S, but not in MM.1RL
Ronco et al. (2010)	Case-control (cell line only)	HSD11β2	Bisulfate sequencing	Choriocarcinoma cell lines	Chorio-carcinoma	Cases: human choriocarcinoma cells, JEG-3 (HTB-36), from The American Type Culture Collection (ATTC, Manassas, VA) exposed to a low dose of cadmium (Cd2 +)Controls: JEG-3 cells not exposed to Cd2 +	•Acute exposure (24 h) to a low dose of Cd2 + (1 μM) induced decreased cortisol production in JEG-3 cells, and was associated with increased HSD11β2 expression and activity •1 μM of Cd2 + induced a reduced methylation index of about 50% in the HSD11β2 promoter, concordant with a decreased expression of the gene, although this change did not reach significance