Research ArticleClinical Studies
Open Access

Evaluation of TET Family Gene Expression and 5-Hydroxymethylcytosine as Potential Epigenetic Markers in Non-small Cell Lung Cancer

AMANI A. ALREHAILI, AMAL F. GHARIB, SALEH ALI ALGHAMDI, AYMAN ALHAZMI, SAAD S. AL-SHEHRI, HOWAIDA M. HAGAG, FOUZEYYAH ALI ALSAEEDI, HAYAA M. ALHUTHALI, NERMIN RAAFAT, RASHA L. ETEWA and WAEL H. ELSAWY
In Vivo January 2023, 37 (1) 445-453; DOI: https://doi.org/10.21873/invivo.13098

Abstract

Background/Aim: DNA methylation is the most studied epigenetic modification in cancer. Ten-eleven translocation enzymes (TET) catalyze the oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in the DNA. In the current research, we aimed to evaluate the role of 5-hmC and TET enzymes in non-small cell lung cancer (NSCLC) patients and their possible association with outcomes. Patients and Methods: ELISA was used to measure the 5-hmC levels in genomic DNA and qRT-PCR was used to evaluate TET1, TET2, and TET3 mRNAs expression levels in NSCLC tissues and their paired normal controls. Results: The levels of 5-hmC were significantly lower in NSCLC tissues than in normal tissues, with a mean ±SD of 0.28±0.37 vs. 1.84±0.58, respectively (t=22.77, p<0.0001), and this reduction was correlated with adverse clinical features. In addition, all TET genes were significantly down-regulated in NSCLC tissues in comparison to their matched normal tissues. The mean±SD level of TET1-mRNA was 38.48±16.38 in NSCLC vs. 80.65±11.25 in normal tissues (t=21.33, p<0.0001), TET2-mRNA level in NSCLC was 5.25±2.78 vs. 9.52±1.01 in normal tissues (t=14.48, p<0.0001), and TET3-mRNA level in NSCLC was 5.21±2.8 vs. 9.51±0.86 in normal tissues (t=14.75, p<0.0001). Downregulation of TET genes was correlated with poor clinical features. Conclusion: 5-HmC levels as well as TET1, TET2, and TET3 mRNA levels were reduced in NSCLC tissues. The reduced levels of 5-hmC and TET mRNAs were associated with adverse clinical features, suggesting that the level of 5-hmC may serve as a valuable prognostic biomarker for NSCLC.

Key Words:

Lung cancer is a main cause of cancer-related death. Non-small cell lung cancer (NSCLC) constitutes about 85% of all lung cancer types (1). In early-stage disease, the 5-year survival is about 58% compared to 15% in advanced stage (2). Therefore, early diagnosis is crucial for improving NSCLC survival rate and the identification of prognostic biomarkers is important (3, 4).

Tumor-suppressor gene (TSG)-associated epigenetic inactivation may lead to tumorigenicity and progression of malignant phenotype in many tumors including NSCLC (5-7). Aberrant DNA methylation of cancer-related gene promoters is considered a significant and early event in carcinogenesis and has gained high interest due to the reversible nature of epigenetic changes (8, 9).

DNA methylation at the fifth position of cytosine is one of the frequent epigenetic changes and has an impact on a variety of cellular functions. Generally, the progression of cancer is linked to 5-methylcytosine (5-mC) at particular genomic locations (10-12). According to the research of Shen et al., the conversion of 5-mC to 5-hydroxymethylcytosine (5-hmC) is essential for epigenetic plasticity (13).

Ten-eleven translocation (TET) enzymes are dioxygenases that convert 5-mC to 5-hmC in DNA by oxidation (14, 15). The oxidation products produce new epigenetic marks and promote further activation of direct or indirect demethylation processes (16). Despite the existence of tissue- and cell-type- specific differences, approximately 5% of cytosines are classified as 5-mC and less than 1% as 5-hmC, in the mammalian cell genome (14). In mammalian genomic DNA, the TET family of dioxygenases can oxidize 5-mC to 5-hmC, 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC). The mammalian thymine DNA glycosylase (TDG) targets and binds 5-fC and 5-caC, which are then repaired to normal cytosine by base excision repair (17). Following conversion to 5-fC and 5-caC, the altered cytosine base is likely to be demethylated via the TDG-dependent process or other possible mechanisms (18). As a result, 5-hmC is considered the main stable epigenetic mark in the DNA demethylation pathway, whereas 5-fC and 5-caC may serve as unstable intermediates (19). 5-hmC is recognized as the sixth base following 5-mC in the mammalian genome (20, 21).

Loss of 5-hmC may be an epigenetic signature of many cancers, with diagnostic and prognostic implications (22). The fact that reduced expression of the three TET genes has been associated with a decrease in the 5-hmC levels in various cancers suggests a plausible mechanism to explain 5-hmC loss in cancer cells (23). In addition, it has shown that reduced levels of 5-hmC are linked to tumorigenesis in genetically engineered mouse models of various types of human malignancies (23). In the current research, we aimed to evaluate the role of 5-hmC and TET enzymes in NSCLC patients and their association with the clinical characteristics of the patients.

Patients and Methods

The current study was conducted at the Medical Biochemistry Department, Faculty of Medicine, Zagazig University. We included 101 patients with NSCLC recruited from the Clinical Oncology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt. All patients were histopathologically confirmed to have NSCLC and underwent surgical excision. Tissue biopsies (tumor tissues and the nearby normal tissues, at least 5cm from the tumor) were obtained from all patients during surgery, flash-frozen in liquid nitrogen and were stored at −80°C until analysis. Clinical staging of all patients was done following the American Joint Committee of Cancer (AJCC) (24). The ethical committees of the Faculty of Medicine, Zagazig University, approved this research (FOMZU:317/2020). All patients had given a written informed consent.

DNA extraction and quantification of 5-hmC by ELISA. Extraction of the Genomic DNA from tumor and normal tissues was done by QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) guided by the directions of the company. We used the Quest 5-hmC™ DNA ELISA Kit (Zymo Research, Irvine, CA, USA) to evaluate 5-hmC according to the company instructions. Using kit controls, we plotted a standard curve and the amount of 5-hmC was calculated as percentage.

Extraction of RNA, cDNA synthesis, and quantitative real-time polymerase chain reaction for TET mRNAs gene expression (qRT-PCR). Total RNA was extracted from the tumor and normal tissues by the miRNeasy Mini Kit (Qiagen) according to the producer’s protocol. The extracted RNA was reverse transcribed into cDNA by the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan) following the manufacturer’s recommendations.

The mRNA expression levels of the TET genes were assessed by qRT-PCR, using the Stratagene, MX3000P quantitative PCR System with the following primers: TET1 forward (F), CCC TTG GAA ATG CCA TAG GAA; TET1 reverse (R), GAG AGC CTG CTG GAA CTG TTG; TET2 F, GGC TGT TGG CCA GAG ACT TA; TET2 R, ATA CCT GTA GGT GTT TGC CTG TTT A; TET3 F, GCC AAC TTC AAC ATA CCC TGG AC; TET3 R, CAC CTG GAT GTG GGA CTG TGTA A; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) F, GCA CCG TCA AGG CTG AGA AC-3′; GAPDH R, TGG TGA AGA CGC CAG TCT CTA; as an internal control (25).

Briefly, the PCR reaction mix (total volume 20 μl) contained 5 μl cDNA, 10 μl Fast SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), and 100 pmol/μl of the primers. The PCR protocol included 40 cycles of 92°C for 20 s and 60°C for 1 min.

The expression was presented as the Δ cycle threshold (ΔCt) value. The relative expression of TET mRNA was calculated using the comparative Ct method and TET values were normalized to the GAPDH mRNA (26). Data were analyzed using MxPro QPCR Software (Agilent Technologies, Santa Clara, CA, USA).

Statistical analysis. Continuous variables are presented as mean±SD. Student’s t-test and one-way analysis of variance (ANOVA) were used to analyse differences in 5-hmC levels between different groups. Spearman’s rank correlation analysis was performed to evaluate the strength and direction of the relationship between groups. For comparisons between tumor and normal tissues, receiver-operator characteristic (ROC) curve analyses were performed on patients’ 5-hmC and TET mRNA levels including histopathology as a gold standard. Statistical significance was defined as a p-value less than 0.05. Statistical analysis was performed using version 9.4 of the SAS software package (SAS Institute, Inc., Cary, NC, USA).

Results

Levels of 5-hmC in NSCLC and their matched normal tissues. The levels of DNA 5-hmC were significantly lower in NSCLC tissues than in their matched normal tissues (0.28±0.37 vs. 1.84±0.58, respectively; t=22.77, p<0.0001) (Figure 1A). Moreover, ROC curve analysis revealed that the 5-hmC levels were highly distinguished between NSCLC tissues and normal tissues with specificity [area under the curve (AUC)=1.000, 95% CI=1.000-1.000, p<0.0001] (Figure 1B). The cutoff value was <1.01 with 100% sensitivity and 98.02% specificity.

Figure 1.
Figure 1.

Levels of 5-hydroxymethylcytosine (5-hmC). (A) ELISA was used to evaluate the levels of 5-hmC (%) in 101 matched pairs of non-small cell lung cancer (NSCLC) and normal tissue specimens (p<0.0001). (B) The area under the receiver operating characteristic (ROC) curve (AUC) of 5-hmC was 1, p<0.0001.

TET expression in NSCLC tissues and their matched normal tissues. We studied the expression levels of TET1, TET2, and TET3 mRNAs in both NSCLC tissues and matched normal tissues. All TET genes were significantly downregulated in NSCLC tissues in comparison to their matched normal tissues. TET1-mRNA level was 38.48±16.38 in NSCLC vs. 80.65±11.25 in normal tissues (t=21.33, p<0.0001), TET2-mRNA level in NSCLC was 5.25±2.78 vs. 9.52±1.01 in normal tissues (t=14.48, p<0.0001), and TET3-mRNA level was 5.21±2.8 in NSCLC vs. 9.51±0.86 in normal tissues (t=14.75, p<0.0001) (Figure 2A, Figure 3A, and Figure 4A).

Figure 2.
Figure 2.

mRNA expression levels of TET1. (A) TET1 mRNA expression levels in 101 matched pairs of non-small cell lung cancer (NSCLC) and normal tissue specimens (p<0.001). (B) The area under the receiver operating characteristic (ROC) curve (AUC) of TET1 was 0.97, p<0.0001.

Figure 3.
Figure 3.

mRNA expression levels of TET2. (A) TET2 mRNA expression levels in 101 matched pairs of non-small cell lung cancer (NSCLC) and normal tissue specimens (p<0.001). (B) The area under the receiver operating characteristic (ROC) curve (AUC) of TET2 was 0.95, p<0.0001.

Figure 4.
Figure 4.

mRNA expression levels of TET3. (A) TET3 mRNA expression levels in 101 matched pairs of non-small cell lung cancer (NSCLC) and normal tissue specimens (p<0.001). (B) The area under the receiver operating characteristic (ROC) curve (AUC) of TET3 was 0.95, p<0.0001.

ROC curves were plotted to evaluate the specificity of TET1, TET2, and TET3 relative mRNA levels in discrimination between NSCLC tissues and normal tissues. The analysis revealed a high specificity of TET1, TET2, and TET3 for discrimination between NSCLC and normal tissues. The AUC for TET1 was 0.97 (p<0.0001, cutoff value >62.31, sensitivity 98.02% and specificity 94.06%), for TET2 was 0.95 (p<0.0001, cutoff value >8.122, sensitivity 95.05%, specificity 84.16%), and for TET3 was 0.95 (p<0.0001, cutoff value 8.195, sensitivity 98.02%, specificity 85.15% specificity) (Figure 2B, Figure 3B, and Figure 4B).

Correlation between 5-hmC levels and TET expression in NSCLC tissues. Spearman correlation analysis revealed a statistically significant positive correlation between 5-hmC levels and mRNA expression levels of TET1 (rs=0.900, p<0.001), TET2 (rs=0.888, p<0.001), and TET3 (rs=0.887, p<0.001), in NSCLC tissues (Figure 5).

Figure 5.
Figure 5.

Spearman’s correlation between 5-hmC and TET1, TET2, and TET3 mRNA levels in non-small cell lung cancer (NSCLC). Correlation between (A) 5-hmC levels and TET1 expression (rS=0.900, p<0.001), (B) 5-hmC levels and TET2 expression (rS=0.888, p<0.001), and (C) 5-hmC levels and TET3 expression (rS=0.887, p<0.001).

Correlation of 5-hmC levels and TET expression with clinical characteristics. Levels of 5-hmC were correlated with clinical characteristics of the patients. Specifically, low levels of 5-hmC were significantly correlated with older age and female sex, as well as with adverse clinical features, such as SCC, high-grade tumors, large tumor size, lymph node involvement, and advanced tumor stage (Table I). Similarly, low mRNA expression levels of TET1, TET2, and TET3 were also significantly associated with all the above-mentioned clinical features (Table II).

View this table:
Table I.

5-hmC levels and patients’ clinical features.

View this table:
Table II.

TET mRNAs expression and patients’ clinical features.

Discussion

Cancer is now widely recognized as a genetic and epigenetic disorder, as certain cancer types often exhibit genetic mutations and repeated changes in the epigenetic environment. In many malignancies, genes encode enzymes, and protein complexes that regulate epigenetic patterns are the most commonly mutated (2). Lung cancer is driven by the accumulation of genetic and epigenetic changes in lung tissue (27). More specifically, genes including p16INK4a, RASSF1A, and FHIT have been found to be usually hypermethylated in NSCLC (28).

DNA methylation is a complicated process and stable epigenetic alteration that allows information to be transmitted from parent to daughter cells. The TET enzymes (TET1, 2, and 3), which are responsible for fine-tuning of demethylation, can reverse DNA methylation by converting 5-mC bases to 5-hydroxymethyl marked bases (29-31). Growing evidence has confirmed the vital role of TET enzymes in controlling gene expression, enhancing cell differentiation, and preventing malignant transformation (32). In the current research, we aimed to evaluate the role of 5-hmC and TET enzymes in NSCLC.

We studied 5-hmC in NSCLC and normal tissues using ELISA and observed a significant suppression of the 5-hmC level in NSCLC tissues compared to the normal controls. Moreover, we plotted a ROC curve for 5-hmC levels, which showed a high specificity of 5-hmC for the discrimination between NSCLC and normal tissues. Several studies have also reported reduced levels of 5-hmC in various malignant tumors (33-35). In a study conducted by Murata et al. in patients with esophageal cancer squamous cell carcinoma (ESCC), 5-hmC was found at lower levels in ESCC tissues compared to normal mucosa (36). Moreover, Wang et al. (37) have revealed global hypomethylation of 5-hmC in lung squamous cell carcinoma tissues compared to paired normal controls, suggesting 5-hmC as a potential biomarker for the early detection of lung cancer.

In the present study, we observed that mRNA expression of TET genes was reduced in NSCLC tissues compared to normal tissues. Our observation was in consistency with the study by Murata et al. in a series of patients with oesophageal cancer, in which only the TET2 level was statistically related to the 5-hmC level while TET1 and TET3 were not correlated to 5-hmC (36). Kudo et al. used immunostaining to examine 5-hmC levels in human cancer tissues (colonic, hepatic, brain, renal, rhabdomyosarcoma, and lung) and normal tissues and discovered that fluorescence patterns denoting 5-hmC were considerably lower in cancerous tissue compared to healthy tissues. The results demonstrated that 5-hmC levels were diminished in colon cancer (72.7%) and stomach cancer (75.0%), confirming the presence of a process that can inhibit the activity of 5-hmC levels in solid malignancies (38). Also, in a series of 546 prostate cancer patients who underwent radical prostatectomy, Storebjerg et al. investigated- 5-hmC by immunohistochemistry of tissue microarray as a possible epigenetic marker. They reported that 5-hmC was significantly reduced in prostatic cancer tissue specimens compared to the normal prostatic tissues (39).

The current study also showed a positive correlation between 5-hmC levels and TET1, TET2, and TET3 mRNA expression in NSCLC tissues. Our observation is in consistency with previous reports in colon adenocarcinoma, lung small cell carcinoma, and other malignancies (33, 34). Putiri et al. have previously reported that the depletion of TET1 in human embryonic carcinoma cells resulted in an increase of 5-mC in promoter elements and a decrease of 5-hmC. At the same time, loss of TET2 and TET3 reduced 5-hmC in a subset of TET1 targets, indicating a functional co-dependence (40). These findings are in line with the known function of TET proteins to catalyze DNA CpG demethylation, allowing normal cells to maintain an appropriate balance between CpG methylation and demethylation (41, 42). Interestingly, it has been shown that CpG methylation-mediated inactivation of the TET1 gene elevated 5-mC levels in many cancer cells of multiple tissue types, suggesting the existence of a DNA methylation feedback loop including CpG methylation and TET1 during tumorigenesis (42).

We noted that reduced 5-hmC levels were associated with adverse clinical and prognostic features in NSCLC, such as older age, female sex, SCC, high-grade tumors, the large size of tumors, lymph node infiltration, and advanced tumor stage. In addition, decreased levels of TET1, TET2, and TET3 in NSCLC were also associated with poor prognostic features, in agreement with previous findings in esophageal cancer (36), renal cell carcinoma (43), brain astrocytoma (44), and urinary bladder cancer (45). Chen et al. showed that 5-hmC was lost in the renal cell carcinoma tissues compared to the matched normal tissues and, moreover, the lower 5-hmC levels were associated with short overall survival (43). In addition, Zhang et al. reported a correlation of reduced 5-hmC with higher pathological grades and shorter survival in a series of patients with WHO grade II diffuse astrocytomas (44). Moreover, Peng et al. conducted a study in patients with urinary bladder cancer. They noted a partial or complete loss of immunoreactivity of 5-hmC malignant tissues compared to normal bladder tissues, while patients with higher 5-hmC immunoreactivity had longer survival than those with lower immunoreactivity. In addition, low 5-hmC immunoreactivity was associated with higher tumor stage and metastasis (45). Misawa et al. (25) have revealed that the levels of 5-hmC were related to the stage of the tumor, and that lower levels of 5-hmC were associated with decreased disease-free survival and local recurrence in head and neck squamous cell carcinoma (HNSCC).

The present study has some limitations. First, our findings were limited to patients having surgery for clinically localized tumors; hence our observations cannot be applied to NSCLC patients with advanced or metastatic disease. Nonetheless, one of the strengths of the current study is that our findings are based on a relatively large consecutive and representative cohort from a single clinical institution, with clinical annotation and follow-up information accessible for all patients. Our study was limited to a relatively large NSCLC cohort from Egypt, and additional independent confirmation is needed. Future validation trials should involve various large NSCLC patient cohorts with long clinical follow-up and more ethnicities.

Conclusion

The levels of 5-hmC were reduced in NSCLC tissues compared to the normal controls and the decreased levels were related to adverse clinical features. In addition, 5-hmC levels were significantly correlated with TET1, TET2, and TET3 mRNA expression. TET genes were downregulated in NSCLC patients and this downregulation was associated with poor prognostic features. Our data imply that 5-hmC is significantly reduced in NSCLC and suggest that loss of 5-hmC may represent a valuable prognostic biomarker for NSCLC.

Acknowledgements

The researchers gratefully thank the financial assistance of Taif University, this study was funded by Taif University Researchers Supporting Project number (TURSP-2020/108), Taif University, Taif, Kingdom of Saudi Arabia.

Footnotes

  • Authors’ Contributions

    Amani A. Alrehaili, Amal F Gharib & Wael H Elsawy conceived and designed experiments; Saleh Ali Alghamdi, Ayman Alhazmi, Saad S. Al-Shehri contributed to the analysis and/or interpretation of data. Howaida M. Hagag, Fouzeyyah Ali Alsaeedi, Nermin Raafat, Rasha L. Etewa drafted the manuscript and Amani A. Alrehaili, Amal F Gharib & Wael H Elsawy revised it for important intellectual content. All Authors have read the manuscript and approved the submission. Hayaa M. Alhuthali had an important role in modifying the reviewers’ notes and the final revision.

  • Conflicts of Interest

    The Authors declare no conflicts of interest

  • Received September 23, 2022.
  • Revision received November 7, 2022.
  • Accepted November 30, 2022.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. Lamichhane SR,
    2. Thachil T,
    3. De Ieso P,
    4. Gee H,
    5. Moss SA and
    6. Milic N
    : Prognostic role of microRNAs in human non-small-cell lung cancer: a systematic review and meta-analysis. Dis Markers 2018: 8309015, 2018. PMID: 30538784. DOI: 10.1155/2018/8309015
    OpenUrlCrossRefPubMed
  2. ↵
    1. Diaz-Lagares A,
    2. Mendez-Gonzalez J,
    3. Hervas D,
    4. Saigi M,
    5. Pajares MJ,
    6. Garcia D,
    7. Crujerias AB,
    8. Pio R,
    9. Montuenga LM,
    10. Zulueta J,
    11. Nadal E,
    12. Rosell A,
    13. Esteller M and
    14. Sandoval J
    : A novel epigenetic signature for early diagnosis in lung cancer. Clin Cancer Res 22(13): 3361-3371, 2016. PMID: 26842235. DOI: 10.1158/1078-0432.CCR-15-2346
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Voigt W,
    2. Manegold C,
    3. Pilz L,
    4. Wu YL,
    5. Müllauer L,
    6. Pirker R,
    7. Filipits M,
    8. Niklinski J,
    9. Petruzelka L and
    10. Prosch H
    : Beyond tissue biopsy: a diagnostic framework to address tumor heterogeneity in lung cancer. Curr Opin Oncol 32(1): 68-77, 2020. PMID: 31714259. DOI: 10.1097/CCO.0000000000000598
    OpenUrlCrossRefPubMed
  4. ↵
    1. Garrido P,
    2. Conde E,
    3. de Castro J,
    4. Gómez-Román JJ,
    5. Felip E,
    6. Pijuan L,
    7. Isla D,
    8. Sanz J,
    9. Paz-Ares L and
    10. López-Ríos F
    : Updated guidelines for predictive biomarker testing in advanced non-small-cell lung cancer: a National Consensus of the Spanish Society of Pathology and the Spanish Society of Medical Oncology. Clin Transl Oncol 22(7): 989-1003, 2020. PMID: 31598903. DOI: 10.1007/s12094-019-02218-4
    OpenUrlCrossRefPubMed
  5. ↵
    1. Llinàs-Arias P and
    2. Esteller M
    : Epigenetic inactivation of tumour suppressor coding and non-coding genes in human cancer: an update. Open Biol 7(9): 170152, 2017. PMID: 28931650. DOI: 10.1098/rsob.170152
    OpenUrlCrossRefPubMed
    1. Kazanets A,
    2. Shorstova T,
    3. Hilmi K,
    4. Marques M and
    5. Witcher M
    : Epigenetic silencing of tumor suppressor genes: Paradigms, puzzles, and potential. Biochim Biophys Acta 1865(2): 275-288, 2016. PMID: 27085853. DOI: 10.1016/j.bbcan.2016.04.001
    OpenUrlCrossRefPubMed
  7. ↵
    1. Misawa K,
    2. Misawa Y,
    3. Kanazawa T,
    4. Mochizuki D,
    5. Imai A,
    6. Endo S,
    7. Carey TE and
    8. Mineta H
    : Epigenetic inactivation of galanin and GALR1/2 is associated with early recurrence in head and neck cancer. Clin Exp Metastasis 33(2): 187-195, 2016. PMID: 26572146. DOI: 10.1007/s10585-015-9768-4
    OpenUrlCrossRefPubMed
  8. ↵
    1. Vaiopoulos AG,
    2. Athanasoula KCh and
    3. Papavassiliou AG
    : Epigenetic modifications in colorectal cancer: molecular insights and therapeutic challenges. Biochim Biophys Acta 1842(7): 971-980, 2014. PMID: 24561654. DOI: 10.1016/j.bbadis.2014.02.006
    OpenUrlCrossRefPubMed
  9. ↵
    1. Nunes SP,
    2. Diniz F,
    3. Moreira-Barbosa C,
    4. Constâncio V,
    5. Silva AV,
    6. Oliveira J,
    7. Soares M,
    8. Paulino S,
    9. Cunha AL,
    10. Rodrigues J,
    11. Antunes L,
    12. Henrique R and
    13. Jerónimo C
    : Subtyping lung cancer using DNA methylation in liquid biopsies. J Clin Med 8(9): 1500, 2019. PMID: 31546933. DOI: 10.3390/jcm8091500
    OpenUrlCrossRefPubMed
  10. ↵
    1. Bhutani N,
    2. Burns DM and
    3. Blau HM
    : DNA demethylation dynamics. Cell 146(6): 866-872, 2011. PMID: 21925312. DOI: 10.1016/j.cell.2011.08.042
    OpenUrlCrossRefPubMed
    1. Esteller M
    : Epigenetics in cancer. N Engl J Med 358(11): 1148-1159, 2008. PMID: 18337604. DOI: 10.1056/NEJMra072067
    OpenUrlCrossRefPubMed
  12. ↵
    1. Jones PA and
    2. Baylin SB
    : The epigenomics of cancer. Cell 128(4): 683-692, 2007. PMID: 17320506. DOI: 10.1016/j.cell.2007.01.029
    OpenUrlCrossRefPubMed
  13. ↵
    1. Shen L and
    2. Zhang Y
    : 5-Hydroxymethylcytosine: generation, fate, and genomic distribution. Curr Opin Cell Biol 25(3): 289-296, 2013. PMID: 23498661. DOI: 10.1016/j.ceb.2013.02.017
    OpenUrlCrossRefPubMed
  14. ↵
    1. An J,
    2. Rao A and
    3. Ko M
    : TET family dioxygenases and DNA demethylation in stem cells and cancers. Exp Mol Med 49(4): e323, 2017. PMID: 28450733. DOI: 10.1038/emm.2017.5
    OpenUrlCrossRefPubMed
  15. ↵
    1. van der Pol Y and
    2. Mouliere F
    : Toward the early detection of cancer by decoding the epigenetic and environmental fingerprints of cell-free DNA. Cancer Cell 36(4): 350-368, 2019. PMID: 31614115. DOI: 10.1016/j.ccell.2019.09.003
    OpenUrlCrossRefPubMed
  16. ↵
    1. Yegnasubramanian S
    : Prostate cancer epigenetics and its clinical implications. Asian J Androl 18(4): 549-558, 2016. PMID: 27212125. DOI: 10.4103/1008-682X.179859
    OpenUrlCrossRefPubMed
  17. ↵
    1. Ito S,
    2. Shen L,
    3. Dai Q,
    4. Wu SC,
    5. Collins LB,
    6. Swenberg JA,
    7. He C and
    8. Zhang Y
    : Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333(6047): 1300-1303, 2011. PMID: 21778364. DOI: 10.1126/science.1210597
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Song CX,
    2. Yi C and
    3. He C
    : Mapping recently identified nucleotide variants in the genome and transcriptome. Nat Biotechnol 30(11): 1107-1116, 2012. PMID: 23138310. DOI: 10.1038/nbt.2398
    OpenUrlCrossRefPubMed
  19. ↵
    1. Wu X and
    2. Zhang Y
    : TET-mediated active DNA demethylation: mechanism, function and beyond. Nat Rev Genet 18(9): 517-534, 2017. PMID: 28555658. DOI: 10.1038/nrg.2017.33
    OpenUrlCrossRefPubMed
  20. ↵
    1. Ye C and
    2. Li L
    : 5-hydroxymethylcytosine: a new insight into epigenetics in cancer. Cancer Biol Ther 15(1): 10-15, 2014. PMID: 24253310. DOI: 10.4161/cbt.27144
    OpenUrlCrossRefPubMed
  21. ↵
    1. Sandanger TM,
    2. Nøst TH,
    3. Guida F,
    4. Rylander C,
    5. Campanella G,
    6. Muller DC,
    7. van Dongen J,
    8. Boomsma DI,
    9. Johansson M,
    10. Vineis P,
    11. Vermeulen R,
    12. Lund E and
    13. Chadeau-Hyam M
    : DNA methylation and associated gene expression in blood prior to lung cancer diagnosis in the Norwegian Women and Cancer cohort. Sci Rep 8(1): 16714, 2018. PMID: 30425263. DOI: 10.1038/s41598-018-34334-6
    OpenUrlCrossRefPubMed
  22. ↵
    1. Qiu L,
    2. Liu F,
    3. Yi S,
    4. Li X,
    5. Liu X,
    6. Xiao C,
    7. Lian CG,
    8. Tu P and
    9. Wang Y
    : Loss of 5-Hydroxymethylcytosine is an epigenetic biomarker in cutaneous T-Cell lymphoma. J Invest Dermatol 138(11): 2388-2397, 2018. PMID: 29803640. DOI: 10.1016/j.jid.2018.05.007
    OpenUrlCrossRefPubMed
  23. ↵
    1. Yang H,
    2. Liu Y,
    3. Bai F,
    4. Zhang JY,
    5. Ma SH,
    6. Liu J,
    7. Xu ZD,
    8. Zhu HG,
    9. Ling ZQ,
    10. Ye D,
    11. Guan KL and
    12. Xiong Y
    : Tumor development is associated with decrease of TET gene expression and 5-methylcytosine hydroxylation. Oncogene 32(5): 663-669, 2013. PMID: 22391558. DOI: 10.1038/onc.2012.67
    OpenUrlCrossRefPubMed
  24. ↵
    1. Amin MB,
    2. Greene FL,
    3. Edge SB,
    4. Compton CC,
    5. Gershenwald JE,
    6. Brookland RK,
    7. Meyer L,
    8. Gress DM,
    9. Byrd DR and
    10. Winchester DP
    : The Eighth Edition AJCC Cancer Staging Manual: Continuing to build a bridge from a population-based to a more “personalized” approach to cancer staging. CA Cancer J Clin 67(2): 93-99, 2017. PMID: 28094848. DOI: 10.3322/caac.21388
    OpenUrlCrossRefPubMed
  25. ↵
    1. Misawa K,
    2. Yamada S,
    3. Mima M,
    4. Nakagawa T,
    5. Kurokawa T,
    6. Imai A,
    7. Mochizuki D,
    8. Morita K,
    9. Ishikawa R,
    10. Endo S and
    11. Misawa Y
    : 5-Hydroxymethylcytosine and ten-eleven translocation dioxygenases in head and neck carcinoma. J Cancer 10(21): 5306-5314, 2019. PMID: 31602281. DOI: 10.7150/jca.34806
    OpenUrlCrossRefPubMed
  26. ↵
    1. Silver N,
    2. Best S,
    3. Jiang J and
    4. Thein SL
    : Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7: 33, 2006. PMID: 17026756. DOI: 10.1186/1471-2199-7-33
    OpenUrlCrossRefPubMed
  27. ↵
    1. Dumitrescu RG
    : Epigenetic markers of early tumor development. Methods Mol Biol 863: 3-14, 2012. PMID: 22359284. DOI: 10.1007/978-1-61779-612-8_1
    OpenUrlCrossRefPubMed
  28. ↵
    1. Langevin SM,
    2. Kratzke RA and
    3. Kelsey KT
    : Epigenetics of lung cancer. Transl Res 165(1): 74-90, 2015. PMID: 24686037. DOI: 10.1016/j.trsl.2014.03.001
    OpenUrlCrossRefPubMed
  29. ↵
    1. Tahiliani M,
    2. Koh KP,
    3. Shen Y,
    4. Pastor WA,
    5. Bandukwala H,
    6. Brudno Y,
    7. Agarwal S,
    8. Iyer LM,
    9. Liu DR,
    10. Aravind L and
    11. Rao A
    : Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324(5929): 930-935, 2009. PMID: 19372391. DOI: 10.1126/science.1170116
    OpenUrlAbstract/FREE Full Text
    1. Ficz G and
    2. Gribben JG
    : Loss of 5-hydroxymethylcytosine in cancer: cause or consequence? Genomics 104(5): 352-357, 2014. PMID: 25179374. DOI: 10.1016/j.ygeno.2014.08.017
    OpenUrlCrossRefPubMed
  31. ↵
    1. Ichimura N,
    2. Shinjo K,
    3. An B,
    4. Shimizu Y,
    5. Yamao K,
    6. Ohka F,
    7. Katsushima K,
    8. Hatanaka A,
    9. Tojo M,
    10. Yamamoto E,
    11. Suzuki H,
    12. Ueda M and
    13. Kondo Y
    : Aberrant TET1 methylation closely associated with CpG island methylator phenotype in colorectal cancer. Cancer Prev Res (Phila) 8(8): 702-711, 2015. PMID: 26063725. DOI: 10.1158/1940-6207.CAPR-14-0306
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Thienpont B,
    2. Steinbacher J,
    3. Zhao H,
    4. D’Anna F,
    5. Kuchnio A,
    6. Ploumakis A,
    7. Ghesquière B,
    8. Van Dyck L,
    9. Boeckx B,
    10. Schoonjans L,
    11. Hermans E,
    12. Amant F,
    13. Kristensen VN,
    14. Peng Koh K,
    15. Mazzone M,
    16. Coleman M,
    17. Carell T,
    18. Carmeliet P and
    19. Lambrechts D
    : Tumour hypoxia causes DNA hypermethylation by reducing TET activity. Nature 537(7618): 63-68, 2016. PMID: 27533040. DOI: 10.1038/nature19081
    OpenUrlCrossRefPubMed
  33. ↵
    1. Ye C and
    2. Li L
    : 5-hydroxymethylcytosine: a new insight into epigenetics in cancer. Cancer Biol Ther 15(1): 10-15, 2014. PMID: 24253310. DOI: 10.4161/cbt.27144
    OpenUrlCrossRefPubMed
  34. ↵
    1. Kroeze LI,
    2. van der Reijden BA and
    3. Jansen JH
    : 5-Hydroxymethylcytosine: An epigenetic mark frequently deregulated in cancer. Biochim Biophys Acta 1855(2): 144-154, 2015. PMID: 25579174. DOI: 10.1016/j.bbcan.2015.01.001
    OpenUrlCrossRefPubMed
  35. ↵
    1. Mariani CJ,
    2. Madzo J,
    3. Moen EL,
    4. Yesilkanal A and
    5. Godley LA
    : Alterations of 5-hydroxymethylcytosine in human cancers. Cancers (Basel) 5(3): 786-814, 2013. PMID: 24202321. DOI: 10.3390/cancers5030786
    OpenUrlCrossRefPubMed
  36. ↵
    1. Murata A,
    2. Baba Y,
    3. Ishimoto T,
    4. Miyake K,
    5. Kosumi K,
    6. Harada K,
    7. Kurashige J,
    8. Iwagami S,
    9. Sakamoto Y,
    10. Miyamoto Y,
    11. Yoshida N,
    12. Yamamoto M,
    13. Oda S,
    14. Watanabe M,
    15. Nakao M and
    16. Baba H
    : TET family proteins and 5-hydroxymethylcytosine in esophageal squamous cell carcinoma. Oncotarget 6(27): 23372-23382, 2015. PMID: 26093090. DOI: 10.18632/oncotarget.4281
    OpenUrlCrossRefPubMed
  37. ↵
    1. Wang Z,
    2. Du M,
    3. Yuan Q,
    4. Guo Y,
    5. Hutchinson JN,
    6. Su L,
    7. Zheng Y,
    8. Wang J,
    9. Mucci LA,
    10. Lin X,
    11. Hou L and
    12. Christiani DC
    : Epigenomic analysis of 5-hydroxymethylcytosine (5hmC) reveals novel DNA methylation markers for lung cancers. Neoplasia 22(3): 154-161, 2020. PMID: 32062069. DOI: 10.1016/j.neo.2020.01.001
    OpenUrlCrossRefPubMed
  38. ↵
    1. Kudo Y,
    2. Tateishi K,
    3. Yamamoto K,
    4. Yamamoto S,
    5. Asaoka Y,
    6. Ijichi H,
    7. Nagae G,
    8. Yoshida H,
    9. Aburatani H and
    10. Koike K
    : Loss of 5-hydroxymethylcytosine is accompanied with malignant cellular transformation. Cancer Sci 103(4): 670-676, 2012. PMID: 22320381. DOI: 10.1111/j.1349-7006.2012.02213.x
    OpenUrlCrossRefPubMed
  39. ↵
    1. Storebjerg TM,
    2. Strand SH,
    3. Høyer S,
    4. Lynnerup AS,
    5. Borre M,
    6. Ørntoft TF and
    7. Sørensen KD
    : Dysregulation and prognostic potential of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) levels in prostate cancer. Clin Epigenetics 10(1): 105, 2018. PMID: 30086793. DOI: 10.1186/s13148-018-0540-x
    OpenUrlCrossRefPubMed
  40. ↵
    1. Putiri EL,
    2. Tiedemann RL,
    3. Thompson JJ,
    4. Liu C,
    5. Ho T,
    6. Choi JH and
    7. Robertson KD
    : Distinct and overlapping control of 5-methylcytosine and 5-hydroxymethylcytosine by the TET proteins in human cancer cells. Genome Biol 15(6): R81, 2014. PMID: 24958354. DOI: 10.1186/gb-2014-15-6-r81
    OpenUrlCrossRefPubMed
  41. ↵
    1. Scourzic L,
    2. Mouly E and
    3. Bernard OA
    : TET proteins and the control of cytosine demethylation in cancer. Genome Med 7(1): 9, 2015. PMID: 25632305. DOI: 10.1186/s13073-015-0134-6
    OpenUrlCrossRefPubMed
  42. ↵
    1. Li L,
    2. Li C,
    3. Mao H,
    4. Du Z,
    5. Chan WY,
    6. Murray P,
    7. Luo B,
    8. Chan AT,
    9. Mok TS,
    10. Chan FK,
    11. Ambinder RF and
    12. Tao Q
    : Corrigendum: Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers. Sci Rep 6: 34435, 2016. PMID: 27708339. DOI: 10.1038/srep34435
    OpenUrlCrossRefPubMed
  43. ↵
    1. Chen K,
    2. Zhang J,
    3. Guo Z,
    4. Ma Q,
    5. Xu Z,
    6. Zhou Y,
    7. Xu Z,
    8. Li Z,
    9. Liu Y,
    10. Ye X,
    11. Li X,
    12. Yuan B,
    13. Ke Y,
    14. He C,
    15. Zhou L,
    16. Liu J and
    17. Ci W
    : Loss of 5-hydroxymethylcytosine is linked to gene body hypermethylation in kidney cancer. Cell Res 26(1): 103-118, 2016. PMID: 26680004. DOI: 10.1038/cr.2015.150
    OpenUrlCrossRefPubMed
  44. ↵
    1. Zhang F,
    2. Liu Y,
    3. Zhang Z,
    4. Li J,
    5. Wan Y,
    6. Zhang L,
    7. Wang Y,
    8. Li X,
    9. Xu Y,
    10. Fu X,
    11. Zhang X,
    12. Zhang M,
    13. Zhang Z,
    14. Zhang J,
    15. Yan Q,
    16. Ye J,
    17. Wang Z,
    18. Chen CD,
    19. Lin W and
    20. Li Q
    : 5-hydroxymethylcytosine loss is associated with poor prognosis for patients with WHO grade II diffuse astrocytomas. Sci Rep 6: 20882, 2016. PMID: 26864347. DOI: 10.1038/srep20882
    OpenUrlCrossRefPubMed
  45. ↵
    1. Peng D,
    2. Ge G,
    3. Gong Y,
    4. Zhan Y,
    5. He S,
    6. Guan B,
    7. Li Y,
    8. Xu Z,
    9. Hao H,
    10. He Z,
    11. Xiong G,
    12. Zhang C,
    13. Shi Y,
    14. Zhou Y,
    15. Ci W,
    16. Li X and
    17. Zhou L
    : Vitamin C increases 5-hydroxymethylcytosine level and inhibits the growth of bladder cancer. Clin Epigenetics 10(1): 94, 2018. PMID: 30005692. DOI: 10.1186/s13148-018-0527-7
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Evaluation of TET Family Gene Expression and 5-Hydroxymethylcytosine as Potential Epigenetic Markers in Non-small Cell Lung Cancer
AMANI A. ALREHAILI, AMAL F. GHARIB, SALEH ALI ALGHAMDI, AYMAN ALHAZMI, SAAD S. AL-SHEHRI, HOWAIDA M. HAGAG, FOUZEYYAH ALI ALSAEEDI, HAYAA M. ALHUTHALI, NERMIN RAAFAT, RASHA L. ETEWA, WAEL H. ELSAWY
In Vivo Jan 2023, 37 (1) 445-453; DOI: 10.21873/invivo.13098
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section

Keywords