**3. Distinct functions of TET1 on tumorigenesis**

### **3.1.** *Tet1* **functions as an oncogene in some cancers**

involving the base excision repair pathway. Second, stepwise, a small percentage (~10%) of the 5-hmC is converted to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), respectively [56, 57]. The 5-fC and 5-caC are finally converted into regular cytosine [58] with the help of Thymine-DNA glycosylase (TDG). Finally, in some tissues such as stem cells and adult neuron cells, high 5-hmC levels could be detected particularly in transcribed regions adjacent to the promoter and enhancers, positively correlating with gene expression. The low turnover rates of 5-hmC in some tissues suggest that besides serving as an intermediate of active demethylation, the stable accumulation of the 5-hmC forms a dynamic 5-hmC landscape to serve as special epigenetic markers, potentially altering the local chromatin structures via recruiting or repelling some special protein components with high affinity to or low even repellent to 5-hmC-harboring DNA [59, 60]. For example, 5-hmC loss has become a hall marker for cancer cells [61–66]. In addition, the TET members are acknowledged as the tumor suppressors as Tet

In mammalian genome, the TET family is consisted of three members, including TET1, TET2, and TET3. While all three TET members could function as hydroxylases for conversion of 5-mC to 5-hmC and further stepwise from 5-hmC to 5-fC and 5-fC to 5-caC, their functions involved in diverse biological pathways are in the development stage and specifically in

Highly expressed in ESCs, PGCs, and inner cell mass of blastocyst, TET1 protein has been proven to be mainly responsible for the initial oxidation of 5-mC to 5-hmC, and to establish the paradoxically dual distinct epigenetic patterns in transcriptional activation and repression in accordance with life processes of growth and development. Alternative splicing mechanism leads to several TET1 isoforms, including the full-length canonical and the short transcripts [69–73]. TET1 expression is regulated by very complicated factors including the reprogramming factors such as Oct3/4, Nanog, and Myc [68, 70] in early embryos, ESCs and PGCs [69], the transcription factors in the differentiated cells, and STAT3/STAT5 in acute myeloid leukemia (AML) [74].

The full length of TET1 protein is believed to have multiple functions in regulation of gene expression. In general, TET1 catalyzes the oxidation of 5-mC to 5-hmC, which serves as an epigenetic marker and intermediate for active demethylation, leading to transcription activation. The more emerging evidence has supported the TET1 conferred transcription activation and repression of its direct target genes [75–77] at the transcriptional level. At the molecular level, the interaction between TET1 and SIN3a facilitates transcription activation of their target genes at the transcription level. More importantly, the interaction has been detected between TET1/ TET2 and E26 transformation-specific or E-twenty-six (ETS) family, one of the largest transcription factor families. For example, ETS variant 2 (ETV2), an ETS family transcription factor, interacts with TET1/TET2 to recruit the demethylases to the Robo4 promoter for demethylationmediated transcription activation during endothelial differentiation. More recently, the Methyl-CpG-binding domain (MBD) protein, such as MBD1, through its CXXC domain recruits TET1 other than TET2 and TET3 to the heterochromatin for oxidation of 5-mC to 5-hmC, whereas the resulting 5-hmC releases the MBD1 from the binding sites by affinity-based displacement [78].

gene mutations or deletions have been identified in some tumor tissues [67].

tissue-dependent manners [25, 68].

190 Chromatin and Epigenetics

*2.2.1. TET1 and regulation of its target gene expression*

Initially, given the mutations and the deletions as predominant variation of TET proteins, particularly TET1, in human cancer genomes, it was accepted that TET1 functions as a tumor suppressor [61, 65, 66]. Indeed, TET1 and TET3 bear the predominant mutations in some tumors including colorectal cancer, melanoma, and cutaneous squamous cell carcinoma [88–90]. However, emerging evidences are connecting the TET1 overexpression and tumorigenesis as well, most likely attributed to activation of cancer-specific oncogenic pathways mediated by TET1 conferred hypomethylation [72, 84] (**Figures 1** and **2**).

*3.1.1. TET1 demethylation associated activation of the members in the oncogenic pathways*

invasion in lung neoplasms, functioning as the potential oncogene [86, 87].

outside of CGIs.

*transcript*

*3.1.2. Hypoxia induced promotion of TET1 expression*

suggesting the oncogenic function of TET1 under hypoxia condition.

*3.1.3. Overexpression of Tet1 mRNA 3'UTRs leads to sequestration of miRNAs, which target the oncogenic transcripts as well, leading to miRNA deficiency to target the oncogenic* 

TET1 overexpression accounts for about 40% of patients with triple-negative breast cancer (TNBC) that belongs to the most hypomethylated cancers observed, leading to about 10% hypomethylation of the queried CGI and activation of oncogenic pathways including PI3K, EGFR, and PDGF. Thus, TET1 seems functioning as a potential oncogene and could serve as a target for intervention therapy [84]. This phenomenon was observed not only in NTBC, but also in MLL-rearranged leukemia where TET1 is believed to activate the downstream oncogenic pathways by its demethylase activity, serving as an oncogene [86]. Additionally, via DNA hypomethylation, TET1 was demonstrated to regulate the expression of MUC4, one member of the mucin (MUC) family and an essential factor for carcinogenesis and tumor

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TET1 functions as an important oncoprotein in acute myeloid leukemia (AML) as evidenced by the high level expression of TET1 in AML, indicating that efficient inhibition of TET1 expression could serve as a powerful strategy for AML therapy. Drug screening led to identification of two compounds NSC-370284 and its structure analogue UC-514321, which repress TET1 transcription by targeting directly to target STAT3/5, TET1 transcriptional activators, suggesting the potential of the compounds targeting the STAT/TET1 for efficient therapy of AML [74]. Full length TET1 (TET1FL) has a CXXC domain that binds to unmethylated CpG islands (CGIs), allowing TET1 to protect CGIs from aberrant methylation and limiting its ability to regulate genes outside of CGIs. An isoform of TET1 (TET1ALT) without CXXC domain but still with catalytic domain is repressed in ES cells while it is activated in embryonic and adult tissues in contrast to TET1FL's expression in ESCs and repression in adult tissues. TET1ALT aberrant activation is detected in breast cancer, uterine and ovarian cancer, and glioblastoma, leading to worse overall survival in these types of cancers. As for the pathogenesis mediated by the TET1ALT isoform, a predominantly activated isoform of TET1 in cancer cells does not protect from CGI methylation but likely mediates dynamic site-specific demethylation

Enhanced expression of TET1 by hypoxia induction has been reported to upregulate cancer cell migration, invasion, and proliferation via the HIF1α signaling pathway in JEG3 cells [100],

Transcription levels of TETs were significantly elevated while the protein levels were not in gastric cancer (GC) tissues compared to the adjacent normal tissues, suggesting the essential role(s) of the endogenous TET transcripts in gastric carcinogenesis and prognosis. Further study showed that overexpression of 5'UTRs, CDs, and 3'UTRs contributed to varied effects in a way that overexpression of TET 3'UTRS promoted GC growth and proliferation. Given that miR-26 targets 3'UTRs of both TET1 and EZH2 mRNAs, overexpression of TET members

**Figure 1.** TET1 functions as a tumor promoter by activation of the oncogenes via demethylation of the methylated promoter regions in the oncogenes. (A) In normal cells, the promoter regions of the oncogenes are usually methylated and therefore silenced. (B) However, in some cells, TET1 is highly expressed and recruited by its interaction partners to the methylated promoter regions of the oncogenes, leading to demethylation and the activation of the oncogenes. Consequently, the oncoproteins initiate tumorigenesis.

**Figure 2.** Sequestration of miR-26 by its target 3'UTRs of Tet1 leads to miR-26 deficiency to target its target Ezh2, an oncogene. (A) In normal cells, due to low level of Tet1 expression, majority of the miR-26 targets to Ezh2 leads to sufficient silencing of the oncogene. (B) In some cancer cells, dramatically enhanced transcription of Tet1 sequestrates the miR-26, conferring the miR-26 deficiency to target its Ezh2 target 3'UTRs. Consequently, miR-26 deficiency to the Ezh2 releases the miRNA-mediated expression repression of the oncogenes, conferring the initiation of tumorigenesis.

### *3.1.1. TET1 demethylation associated activation of the members in the oncogenic pathways*

TET1 overexpression accounts for about 40% of patients with triple-negative breast cancer (TNBC) that belongs to the most hypomethylated cancers observed, leading to about 10% hypomethylation of the queried CGI and activation of oncogenic pathways including PI3K, EGFR, and PDGF. Thus, TET1 seems functioning as a potential oncogene and could serve as a target for intervention therapy [84]. This phenomenon was observed not only in NTBC, but also in MLL-rearranged leukemia where TET1 is believed to activate the downstream oncogenic pathways by its demethylase activity, serving as an oncogene [86]. Additionally, via DNA hypomethylation, TET1 was demonstrated to regulate the expression of MUC4, one member of the mucin (MUC) family and an essential factor for carcinogenesis and tumor invasion in lung neoplasms, functioning as the potential oncogene [86, 87].

TET1 functions as an important oncoprotein in acute myeloid leukemia (AML) as evidenced by the high level expression of TET1 in AML, indicating that efficient inhibition of TET1 expression could serve as a powerful strategy for AML therapy. Drug screening led to identification of two compounds NSC-370284 and its structure analogue UC-514321, which repress TET1 transcription by targeting directly to target STAT3/5, TET1 transcriptional activators, suggesting the potential of the compounds targeting the STAT/TET1 for efficient therapy of AML [74].

Full length TET1 (TET1FL) has a CXXC domain that binds to unmethylated CpG islands (CGIs), allowing TET1 to protect CGIs from aberrant methylation and limiting its ability to regulate genes outside of CGIs. An isoform of TET1 (TET1ALT) without CXXC domain but still with catalytic domain is repressed in ES cells while it is activated in embryonic and adult tissues in contrast to TET1FL's expression in ESCs and repression in adult tissues. TET1ALT aberrant activation is detected in breast cancer, uterine and ovarian cancer, and glioblastoma, leading to worse overall survival in these types of cancers. As for the pathogenesis mediated by the TET1ALT isoform, a predominantly activated isoform of TET1 in cancer cells does not protect from CGI methylation but likely mediates dynamic site-specific demethylation outside of CGIs.

#### *3.1.2. Hypoxia induced promotion of TET1 expression*

**Figure 2.** Sequestration of miR-26 by its target 3'UTRs of Tet1 leads to miR-26 deficiency to target its target Ezh2, an oncogene. (A) In normal cells, due to low level of Tet1 expression, majority of the miR-26 targets to Ezh2 leads to sufficient silencing of the oncogene. (B) In some cancer cells, dramatically enhanced transcription of Tet1 sequestrates the miR-26, conferring the miR-26 deficiency to target its Ezh2 target 3'UTRs. Consequently, miR-26 deficiency to the Ezh2 releases the miRNA-mediated expression repression of the oncogenes, conferring the initiation of tumorigenesis.

**Figure 1.** TET1 functions as a tumor promoter by activation of the oncogenes via demethylation of the methylated promoter regions in the oncogenes. (A) In normal cells, the promoter regions of the oncogenes are usually methylated and therefore silenced. (B) However, in some cells, TET1 is highly expressed and recruited by its interaction partners to the methylated promoter regions of the oncogenes, leading to demethylation and the activation of the oncogenes.

Consequently, the oncoproteins initiate tumorigenesis.

192 Chromatin and Epigenetics

Enhanced expression of TET1 by hypoxia induction has been reported to upregulate cancer cell migration, invasion, and proliferation via the HIF1α signaling pathway in JEG3 cells [100], suggesting the oncogenic function of TET1 under hypoxia condition.

## *3.1.3. Overexpression of Tet1 mRNA 3'UTRs leads to sequestration of miRNAs, which target the oncogenic transcripts as well, leading to miRNA deficiency to target the oncogenic transcript*

Transcription levels of TETs were significantly elevated while the protein levels were not in gastric cancer (GC) tissues compared to the adjacent normal tissues, suggesting the essential role(s) of the endogenous TET transcripts in gastric carcinogenesis and prognosis. Further study showed that overexpression of 5'UTRs, CDs, and 3'UTRs contributed to varied effects in a way that overexpression of TET 3'UTRS promoted GC growth and proliferation. Given that miR-26 targets 3'UTRs of both TET1 and EZH2 mRNAs, overexpression of TET members mRNA sequestrates miR-26 competitively and leads to release of the miR-26 mediated repression of EZH2. Thus, activation of EZH2 expression facilitates gastric carcinogenesis and progression [87] (**Figure 2**).

of colony formation, cell migration, and invasion by ectopic expression of TET1 in SKOV3 and OVCAR3 cells. The potential mechanism is the TET1 conferred demethylation and the consequent activation of the expression of two key proteins SFRP2 and DKK1 in the canonical Wnt/β-

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TET1 is identified as a key tumor suppressor player in ovarian cancer cell lines as well by demethylating a CpG site within the Ras association domain family member 5 (RASSF5) promoter to enhance expression of the RASSF5, leading to the growth inhibition of ovarian

More evidences show that EGFR-mediated TET1 repression induces silencing of tumor suppressors in cancer cells such as lung adenocarcinomas and glioblastomas. If only the oncogenic EGFR expression is inhibited, TET1 could bind to promoters of the tumor suppressors to activate their expression via DNA demethylation. TET1 overexpression inhibits lung and glioblastoma tumor growth, and vice versa, in agreement with the significant decrease in TET1 expression or TET1 cytoplasmic localization in the majority of lung cancer samples. Thus, it is plausible to speculate that TET1 may serve as the therapeutic target for oncogenic EGFR-induced lung cancers and glioblastomas [93]. However, Lai et al. could not draw the same conclusion in human NSCLC patient samples. They did not detect the EGFR-mediated TET1 silencing, but rather observed the significant elevation of the TET1 expression levels in patient samples with EGFR mutations, suggesting the inconclusiveness in EGFR-mediated TET1 silencing among

Eicosapentaenoic acid (EPA), one of the major polyunsaturated fatty acids, could enhance the formation of PPARγ-RXRα-TET1 to recruit TET1 to a hypermethylated CpG island on the p21 gene for rapid demethylation and consequent expression of p21Waf1/Cip1, leading to inhibition of cancer cell-cycle progression in hepatocarcinoma cells. This suggests the bridge requirement for TET1 exerting the anti-tumor function and potential of EPA for solid tumor

Loss of 5-hydroxymethylcytosine (5 hmC) caused by TET1 dysfunction could induce tumor initiation and enhance malignancy by promoting cancer cell growth, migration, and invasion in DLD1 colon cancer cells mediated by EZH2 [96]. With loss of TET1, EZH2 repression is released, but H3K27 demethylase UTX-1 expression is repressed, enhancing histone H3K27 tri-methylation and consequently repressing the target gene E-cadherin (DH1). Accordingly, even at the condition of TET1 deficiency, either the H3K27 demethylase UTX-1 overexpression or EZH2 depletion both could enhance H3K27 demethylation at CDH1 promoter, thereby impeding EMT and tumor invasion. Likewise, either EZH2 overexpression or UTX-1 depletion both could promote EMT and tumor metastasis in DLD1 cells. Thus, these results elucidate regulation interplay among TET1, E-cadherin, and EZH2 and indicate the critical mediator role the EZH2 plays in the E-cadherin repression and tumor progression [95].

Some miRNAs are identified to be involved in regulation of cancer progression or repression, and one of the mechanisms refers to the oncogenic miRNA-mediated TET1 repression

catenin signaling pathway, associated with inhibition of EMT and metastasis [101].

the cellular and animal models and human lung cancer patients [94].

*3.2.2. TET1 silencing and loss of 5-hmC induces initiation of tumors*

*3.2.3. miRNA-mediated repression of TET1 expression*

cancer cells [102].

therapy such as live cancer [97].

### **3.2. TET1 serves as a tumor suppressor**

The pathogenic contributions the TET members made in various human cancers by functioning as tumor suppressors or promoters have been proven to be versatile. The hypermethylationbased transcriptional silencing of TET1 is frequently detected in non-Hodgkin B cell lymphoma (B-NHL), suggesting TET1 as a tumor suppressor of hematopoietic malignancy [91]. Similarly, TET1 is downregulated upon NF-κB activation in multiple cancers including basal-like breast cancer (BLBC), melanoma, lung, and thyroid cancers, demonstrating that TET1 is the tumor suppressor that relies on involvement of the immune system [92].
