Table 1.

Summary results of selected studies on DNA hydroxymethylation and three major smoking-associated cancers (i.e., lung, bladder, and colorectal cancers).

Study	Cancer Type	Samples	Method(s)	Key Findings	Ref.
Li et al.	Lung	Tumors from lung cancer patients and adjacent normal tissues (n = 3)	oxBS-seq and RNA-seq	-5-hmC was significantly enriched in promoters, gene bodies, and transcription termination regions. -There was strong positive correlation between 5-hmC and gene expression levels. -The genomic distribution of 5-hmC highly corresponded with the active histone mark H3K4me1.	[178]
Wang et al.	Lung	Lung squamous cell carcinomas and adjacent normal tissues (n = 8)	TAB-EPIC	-Global loss of 5-hmC together with enrichment of 5-hmC in CpG islands and gene upstreams was detected in tumors as compared to normal tissues. -The differentially hydroxymethylated genes converged at pathways involved in cellular process, biological regulation, and metabolic process.	[179]
Song et al.	Lung	Plasma cfDNA from lung cancer patients (n = 15) and healthy controls (n = 8)	hMe-Seal	-Depletion of global 5-hmC levels was detected in cfDNA of lung cancer patients as compared to healthy controls. -The extent of reduction in global 5-hmC levels in patients’ cfDNA increased progressively as the disease advanced from early-stage non-metastatic to late-stage metastatic lung cancer.	[180]
Zhang et al.	Lung	Plasma cfDNA from non-small-cell lung cancer patients (n = 66) and healthy controls (n = 67)	hMe-Seal	-Significantly increased 5-hmC levels were found in gene bodies and promoter regions in cfDNA of lung cancer patients as compared to controls.	[181]
Forloni et al.	Lung	Lung adenocarcinoma cell lines	shRNA knockdown assays, soft agar assay, and tumorigenicity in nude mice	-Oncogenic EGFR was shown to silence multiple tumor suppressors in lung cancer cell lines via transcriptional downregulation of TET1 by the C/EBPα transcription factor.	[182]
Peng et al.	Bladder	Bladder tumors and matching normal tissues from urothelial carcinoma patients (n = 135) andbladder cancer cell lines and controls	hMeDIP-seq, IHC, IDB, MTS cell proliferation assay, apoptosis assay, colony formation assay, and xenograft mouse tumorigenicity	-Global loss of 5-hmC was detected in tumor tissues from bladder cancer patients as well as bladder cancer cell lines as compared to controls. -5-hmC levels were significantly decreased within genes or in the regions 2 kb up- or downstream of the genes in bladder tumors as compared to controls. -Depletion of global 5-hmC levels in bladder cancer patients correlated with higher tumor stage, lymphatic metastasis, and shorter overall survival. -The differentially hydroxymethylated genes converged on molecular pathways involved in cancer. -In vitro treatment of bladder cancer cell lines with vitamin C resulted in increased 5-hmC levels and inhibition of malignant phenotypes. -In vivo treatment of mice with vitamin C by i.p. injection resulted in increased 5-hmC levels, reduced tumor growth, and decreased tumor burden.	[195]
Munari et al.	Bladder	Tumors and adjacent benign tissues from patients with urothelial cell carcinoma of the bladder (n = 55)	IHC	-Global 5-hmC levels were significantly reduced in tumors from patients as compared to control tissues. -The reduction in global 5-hmC levels was not different between superficial tumors and invasive tumors. -The extent of reduction in global 5-hmC levels was not correlated to tumor grade or stage, or patients’ prognosis.	[196]
Zhu et al.	Bladder	Bladder cancer cell lines	shRNA knockdown assays, rescue experiments for gene expression, MSP, RIP, RT-PCR, Western blot, cell migration, invasion, and lung metastasis assays	-The TET1/USP28/CD44/RhoGDIβ pathway was identified as the regulator of the oncogenic activities of ATG7 for stem-like property, invasion, and lung metastasis of human bladder cancer cells.	[197]
Hu et al.	Bladder	Bladder cancer cell lines	RIP, ChIP, RT-qPCR, Western blot, and cell proliferation, migration, and invasion assays	-The XIST-TET1-p53 regulatory network was identified as a regulator of cell proliferation, migration, and apoptosis in bladder cancer cells.	[198]
Dziaman et al.	Colorectal	Tumors and adjacent normal colonic tissues from patients with CRC (n = 97), colon samples from AD (n = 39), and IBD patients (n = 49)	2D-UPLC-MS/MS, RT-qPCR, and IHC	-5-hmC levels were significantly lower in tumor tissues from CRC patients, followed by samples from AD and IDB patients as compared to normal colonic tissues. -Whereas early stage (‘A’) tumors had significant reduction in 5-hmC content, no further decrease in 5-hmC levels was found in advanced stage tumors (‘B–D’). -TET1 and TET2 mRNA expressions were significantly decreased in CRC and AD samples as compared to normal colon samples. -Reduced expressions of TET1 and TET2 proteins were observed in CRC samples as compared to normal colonic tissues. -No changes in expression of TET3 at mRNA or protein level were observed in the analyzed samples. -The levels of biomarker of oxidative DNA damage (8-oxodG) were significantly increased in samples from IBD and AD patients as compared to samples from CRC patients and normal colonic tissues.	[154]
Chapman et al.	Colorectal	Colon adenocarcinoma cell lines and primary human colonocytes	hMe-Seal, IDB, TAB-seq, RNA-seq, and shRNA knockdown assays	-Global 5-hmC levels increased during differentiation of colon cancer cells. -5-hmC levels progressively increased at CpG islands, CpG shores, promoters, and gene bodies in colon cancer cells during differentiation (days 0, 4, 12, and 15). -The 5-hmC enriched regions during differentiation of colon cancer cells localized to genes involved in epithelial barrier function (i.e., focal adhesion, adherens junctions, regulation of actin cytoskeleton, and endocytosis). -Genes associated with MAPK signaling pathway were induced during differentiation of colon cancer cells, whereas numerous metabolic and disease-associated pathways were repressed, simultaneously. -The upregulated genes had higher 5-hmC content than genes that were downregulated or exhibited unchanged expression during differentiation of colon cancer cells. -TET1 expression was induced during differentiation of colon cancer cells. -TET1 knockdown in colonocytes changed the expression of genes coding for proteins targeted to cell membrane and extracellular space, thus inhibiting barrier formation. -Gene-specific 5-hmC changes were directly correlated to expression changes in the corresponding genes in colon cancer tissues.	[207]
Li et al.	Colorectal	Tumors and adjacent normal tissues and plasma cfDNAs from CRC patients (n = 80) and plasma cfDNAs from healthy controls (n = 90)	CE–ESI–MS and RNA-seq	-Global loss of 5-hmC levels was detected in both tumor tissues and cfDNA from CRC patients, with the former showing more pronounced reduction in 5-hmC content. -5-hmC was enriched within gene bodies and DNase I sensitive sites in cfDNA of cancer patients as compared to controls, whereas it was depleted at TSS, CpG islands, and TF binding sites relative to the flanking regions. The 5-hmC-enriched regions were marked by permissive histone modifications (H3K27ac, H3K4me1, and H3K9me1), whereas the depleted 5-hmC regions were marked by the repressive modification H3K9me3. -In tumor samples, changes in 5-hmC levels in gene bodies were significantly corelated to expression changes in the corresponding genes. -The differentially hydroxymethylated genes in tumors or cfDNA from CRC patients were enriched in cancer- and metastasis-related pathways. -A classifier, derived from differentially hydroxymethylated loci in cfDNA and gDNA in CRC patients, predicted disease status, with high sensitivity (80–88%) and specificity (83–100%) in independent subpopulations of CRC patients and healthy controls. This classifier also performed better than the conventional biomarkers and epidemiological risk factors when predicting colorectal cancer.	[209]
Rawluzko-Wieczorek et al.	Colorectal	Primary tumors and histopathologically unchanged tissues from CRC patients (n = 113)	RT-qPCR and bisulfite sequencing	-TET1, TET2, and TET3 transcript levels were significantly reduced in tumors as compared to control tissues. -High TET2 mRNA levels in histopathologically normal tissues from CRC patients associated with favorable overall survival and disease-free survival. -In tumor samples, promoter hypermethylation was found only in TET1 (12/113 = 10.6%) but not in TET2 or TET3.	[210]
Neri et al.	Colorectal	Primary tumors and adjacent healthy tissues from colon cancer patients (n = 8) and normal epithelial colon cells and CRC cell lines	IDB, RT-qPCR, shRNA knockdown assays, and xenograft mouse tumorigenicity	-TET1 transcript and 5-hmC levels were both reduced in tumor tissues as compared to controls. -Downregulation of TET1 was independent of patients’ tumor stage and histopathological grade. -No detectable levels of TET1 expression or 5-hmC were found in CRC cells lines. -Silencing of TET1 in normal epithelial colon cells resulted in increased cell proliferation, whereas re-expression of TET1 in colon cancer cells markedly increased 5-hmC levels and suppressed growth rate. -Mice injected with DOX-induced TET1-expressing colon cancer cells developed significantly smaller tumors (both in size and weight) than counterpart mice injected with non-induced TET1 cells. -Functional studies identified DNA hydroxymethylation mediated by TET1-controlled WNT signaling as a key player of tumor growth in colon cancer.	[211]

Given the extensive body of literature, we have selected illustrative examples of studies on lung, bladder, and colorectal cancers, which are three major smoking-associated cancers. oxBS-seq = oxidative bisulfite sequencing; TAB-EPIC = TET-assisted bisulfite–Infinium Methylation EPIC BeadChip array; cfDNA = cell-free DNA; hMe-Seal = selective chemical labeling of 5-hmC with biotin for genome-wide detection; shRNA = small/short hairpin RNA; EGFR = epidermal growth factor receptor; IHC = immunohistochemistry; IDB = immunodot blot assays; hMeDIP-seq = hydroxymethylated DNA immunoprecipitation with deep sequencing; MSP = methylation-specific polymerase chain reaction; RIP = RNA immunoprecipitation; RT-qPCR = reverse-transcription quantitative polymerase chain reaction; ATG7 = autophagy-related gene 7; ChIP = chromatin immunoprecipitation; XIST = X-inactive specific transcript; CRC = colorectal cancer; AD = benign polyps/colon adenomas; IBD = inflammatory bowel disease; 2D-UPLC-MS/MS = two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry; 8-oxodG = 8-oxo-7,8-dihydro-2′-deoxyguanosine; TAB-seq = TET-assisted bisulfite sequencing; MAPK = mitogen-activated protein kinase; CE–ESI–MS = capillary electrophoresis–electrospray ionization–mass spectrometry; DOX = doxycycline.