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. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: J Invest Dermatol. 2021 Oct 23;142(5):1270–1279.e2. doi: 10.1016/j.jid.2021.09.026

Regulation of 5-hydroxymethylcytosine by TET2 contributes to Squamous Cell Carcinoma tumorigenesis

Rafik Boudra 1,2,#, Yvon Woappi 1,2,#, Diana Wang 3,5, Shuyun Xu 3, Michael Wells 3, Chrysalyne D Schmults 1,2, Christine G Lian 3,4, Matthew R Ramsey 1,2,*
PMCID: PMC9033889  NIHMSID: NIHMS1750675  PMID: 34695415

Abstract

DNA methylation is a key regulatory event controlling a variety of physiological processes and can have dramatic effects on gene transcription. Methylated Cytosine (5mC) can be oxidized by the TET family of enzymes to 5-hydroxymethylcytosine (5-hmC), a key intermediate in the demethylation cycle, and 5-hmC levels are reduced in malignancies such as acute myeloid leukemia and melanoma. We constructed a tissue microarray of human cutaneous Squamous Cell Carcinoma (Squamous Cell Carcinoma) tumors and found a global reduction in 5-hmC levels compared to adjacent skin. Using a murine K14-CreER system, we have found that loss of Tet2 promotes carcinogen-induced Squamous Cell Carcinoma and cooperates with loss of Tp53 to drive spontaneous Squamous Cell Carcinoma tumors in epithelial tissues. Analysis of changes in 5-hmC and gene expression following loss of Tet2 in the epidermis revealed focal alterations in 5-hmC levels and an increase in Hair Follicle Transient Amplifying Cell genes along with a reduction in epidermal differentiation genes. These results demonstrate a role for TET2 in epidermal lineage specification, consistent with reported roles for TET enzymes in controlling lineage commitment in hematopoietic stem cells and ES cells and establish TET2 as a bone fide tumor suppressor in Squamous Cell Carcinoma.

Graphical Abstract

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INTRODUCTION

Covalent modification of Cytosine residues by methylation is an important regulator of gene transcription (Kohli and Zhang, 2013, Pastor et al., 2013). The DNA methylation-demethylation cycle proceeds through a series of intermediates, with 5-methylcytosine (5-mC) being converted first to 5-hydroxymethylcytosine (5-hmC), then to 5-formylcytosine, 5-carboxycytosine, and finally unmethylated cytosine (Kohli and Zhang, 2013, Pastor et al., 2013). Conversion of 5-mC to 5-hmC is carried out by the ten-eleven translocation (TET) family of enzymes (TET1/2/3) (Ito et al., 2010, Ito et al., 2011, Tahiliani et al., 2009), and these enzymes can also contribute to subsequent oxidation steps (He et al., 2011, Ito et al., 2011). Oxidation of 5-mC to 5-hmC is essential for proper differentiation, as ES cells from mice lacking Tet1/2/3 are unable to develop into all germ layers (Ficz et al., 2011, Ito et al., 2010). Regulation of DNA methylation is also important for maintaining stem cells pools in adult tissues, such as the epidermis (Li et al., 2020) hematopoietic cells (Moran-Crusio et al., 2011, Quivoron et al., 2011).

Reductions in 5-hmC are found in many tumor types (Haffner et al., 2011), including acute myeloid leukemia (AML) (Konstandin et al., 2011), melanoma (Bonvin et al., 2019, Lian et al., 2012), Oral Squamous Cell Carcinoma (SCC) (Cuevas-Nunez et al., 2018, Jawert et al., 2013) and Esophageal SCC (Murata et al., 2015) compared to normal cells, suggesting global dysregulation of the methylation cycle in cancer. Studies in Acute Myeloid Leukemia (AML) have suggested that TET proteins may function as tumor suppressors responsible for the loss of 5-hmC (Delhommeau et al., 2009, Jankowska et al., 2009, Moran-Crusio et al., 2011, Tefferi et al., 2009). Across all tumor types, TET2 is mutated at much higher frequency than TET1 or TET3 (Jeschke et al., 2016), and TET2 inactivating mutations have been correlated with reductions in 5-hmC levels in AML (Ko et al., 2010). Further support for a key role for TET2 in tumorigenesis comes from studies in melanoma, where overexpression of TET2 in melanoma cells with low 5-hmC can suppress tumor growth in vivo (Lian et al., 2012). This tumor suppressor ability depends on the catalytic activity of TET2 which is also required to re-establish the 5-hmC epigenetic landscape (Lian et al., 2012), suggesting that 5-hmC loss in melanoma is indeed due to reduced TET activity. In addition, expression of TET2, but not TET1 or TET3 is significantly correlated with 5-hmC levels in Esophageal SCC (Murata et al., 2015). However, the role of Tet2 loss in causing 5-hmC reductions and promoting SCC tumorigenesis has not been tested.

RESULTS

Global 5-hydroxymethylcytosine levels are reduced in Human Cutaneous SCC

The skin is the most frequent site of SCC tumor formation, but the level of 5-hmC in cutaneous SCC has not been examined. We have constructed a human tissue microarray containing 98 cutaneous primary SCC samples and 83 adjacent normal skin samples. These include T1 (N=10), T2a (N=20), and T2b (N=68) tumors evaluated according to the BWH staging system (Jambusaria-Pahlajani et al., 2013, Karia et al., 2014). We have performed Immunohistochemistry on the tissue microarray using antibodies against 5-hmC, and quantified results by counting number of positive tumor cells and scoring for relative intensity of staining (Figure 1a). Consistent with previous results in melanoma (Lian et al., 2012), oral SCC (Cuevas-Nunez et al., 2018), and esophageal SCC (Murata et al., 2015), we have found that global 5-hmC levels are reduced in cutaneous SCC compared to normal skin, however, we saw no clear association between reductions in 5-hmC and tumor stage (Figure 1b). TET family enzymes have been found to be mutated in many different tumor types (Jeschke et al., 2016), and we sought to examine alterations in SCC across tissues (Han et al., 2018, Hoadley et al., 2018, Li et al., 2015, Lin et al., 2014, Pickering et al., 2014, Song et al., 2014) using the cBio cancer genomics portal (Cerami et al., 2012). While TET2 is mutated at much higher frequency than TET1 or TET3 across all tumor types (Jeschke et al., 2016), genomic alterations in TET1, TET2, and TET3 were found in 3–4% of SCC tumors, and some samples had alterations in more than one TET gene (Figure 1c). Aggressive and metastatic cutaneous SCC tumors (Li et al., 2015, Pickering et al., 2014) had the highest frequency of TET gene alterations, and alterations reported were mostly point mutations (Figure 1d). Previous studies in Esophageal SCC demonstrated loss of 5-hmC in tumors, which correlated strongly with reductions in TET2, but not with TET1 or TET3 mRNA levels (Murata et al., 2015). Consistent with this data, only point mutations and deletions were found in TET2 across SCC tumors from all sites, while TET1 and TET3 were found to be amplified in some tumors. We therefore hypothesized that TET2 may play an important tumor suppressor role in SCC.

Figure 1: Altered regulation of 5-hmC in human Squamous Cell Carcinoma.

Figure 1:

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(a) Representative samples of immunohistochemical staining of global levels of 5-hmC (brown) in the human Cutaneous SCC tumors and normal skin from the Tissue Microarray. Numbers indicate scoring of percentage and intensity. Scale bar = 100μm

(b) Quantification of tumor-adjacent skin (n=83) and stage T1 (N=10), T2a (N=20), and T2b (N=68) Cutaneous SCC tumors. *** p<0.001 assessed by Student’s unpaired t-test.

(c) Percentage and type of genomic alterations in TET1, TET2, and TET3 reported in SCC tumors across tissues (Han et al., 2018; Hoadley et al., 2018; Li et al., 2015. Lin et al., 2014; Pickering et al., 2014; Song et al., 2014) assessed using the cBio cancer genomics portal (Cerami et al., 2012). Only samples with alterations are shown.

(d) Absolute counts (top) and frequency (bottom) of genomic alterations in TET1, TET2, and TET3 found in the indicated SCC tumor type.

5-hmC, 5-hydroxymethylcytosine; CNA, copy number alterations; cSCC, cutaneous Squamous Cell Carcinoma; NSCLC, non-small cell lung cancer; SCC, Squamous Cell Carcinoma; TMA. tissue microarray.

Tet2 has tumor suppressor functions in Squamous Cell Carcinoma

Stratified epithelial tissues function as a barrier from the external environment, and a major driver of human SCC is exposure to carcinogens (Dotto and Rustgi, 2016). We have previously found that oral SCC tumors demonstrate loss of 5-hmC (Cuevas-Nunez et al., 2018), but it was unclear if Tet2 could function as a tumor suppressor in this context. We employed an oral carcinogenesis model where mice were subjected to 100μg/mL 4-Nitroquinoline-N-oxide (4-NQO) in their drinking water for 8 consecutive weeks, then analyzed 16 weeks later for tumor formation (Kanojia and Vaidya, 2006). K14-CreER mice, which express an inducible Cre recombinase in basal keratinocytes (Vasioukhin et al., 1999) were crossed to Tet2 conditional knockout mice (Tet2L/L) (Moran-Crusio et al., 2011). Treatment with 100mg/kg tamoxifen for 5 consecutive days resulted in dramatically reduced Tet2 protein and a modest reduction in 5-hmC levels in the oral mucosa of K14-CreER Tet2L/L mice compared to K14-CreER Tet2+/+ mice (Figure 2a). Tamoxifen-treated K14-CreER Tet2L/L (n=16) and Tet2L/L (n=5) mice were then given 4-NQO in their drinking water. In addition, K14-CreER Tet2L/L (n=6) and Tet2L/L (n=3) mice which did not receive tamoxifen were also given 4-NQO in their drinking water. Following treatment with 4-NQO, mice developed dysplasia, papillomas, and SCC (Figure 2b), and oral SCC tumors demonstrated loss of Tet2 protein (Figure 2c). We observed a significant increase in the number of total lesions upon loss of Tet2 in the oral mucosa (Figure 2d). In addition, a significantly higher percentage of lesions that formed in Tet2−/− mice were oral SCC tumors while lesions that developed in Tet2+/+ mice were mostly dysplasia and benign papillomas (Figure 2e).

Figure 2. Tet2 functions as a tumor suppressor in carcinogen-induced oral SCC.

Figure 2.

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(a) Immunohistochemical staining of Tet2 (left) and 5-hmC (right) in the tongue of mice of indicated genotype following tamoxifen treatment. Scale bar = 50μm

(b) Representative images of each tumor grade in the tongue following 4-NQO treatment. Note that invasive SCC tumors were designated grade IV. Scale bar = 500μm

(c) Histology (left) and Immunohistochemical staining of Tet2 (right) in an SCC tumor from a K14-CreER Tet2L/L mouse treated with tamoxifen and 4-NQO. Scale bar = 100μm

(d) Quantification of oral lesions from Tet2+/+ (Tet2L/L + tamoxifen, n=5; Tet2L/L + vehicle, n=3; K14-CreER Tet2L/L + vehicle, n=6) or Tet2−/− (K14-CreER Tet2L/L + tamoxifen, n=15) mice following treatment with 4-NQO. * p<0.05 by Mann-Whitney test.

(e) Quantification of highest lesion grade in Tet2+/+ (Tet2L/L + tamoxifen, n=5; Tet2L/L + vehicle, n=3; K14-CreER Tet2L/L + vehicle, n=6) or Tet2−/− (K14-CreER Tet2L/L + tamoxifen, n=16) mice following treatment with 4-NQO. * p<0.05 by Fisher’s Exact test

4-NQO, 4-nitroquinoline-N-oxide; 5-hmC, 5-hydroxymethylcytosine; SCC, Squamous Cell Carcinoma.

Germline loss of Tet2 leads to myeloid transformation with long latency (Moran-Crusio et al., 2011) and no phenotypes were reported in stratified epithelial tissues despite expression of TET2 in the oral cavity and the skin (Figures 2a, 3a, and b) suggesting the need for cooperating events to drive tumorigenesis. The TP53 tumor suppressor is inactivated in most SCC tumors (Cancer Genome Atlas, 2015, Cancer Genome Atlas Research, 2012), and can promote genome stability (Lane, 1992) and help regulate DNA methylation (Tovy et al., 2017). We hypothesized that loss of Tp53 would cooperate with Tet2 loss to drive SCC tumorigenesis. We crossed p53Flox mice (Marino et al., 2000) with K14-CreER Tet2L/L mice and treated 6-week-old mice with 100mg/kg tamoxifen for 5 consecutive days resulting in loss of Tet2 protein and reduced 5-hmC levels in the epidermis (Figure 3b). As expected, no tumors developed in control Tet2+/+, L/+, or L/L p53+/+, L/+, or L/L mice lacking the K14-CreER transgene following tamoxifen treatment (Figure 3c). Consistent with studies of germline Tet2−/− mice (Moran-Crusio et al., 2011, Quivoron et al., 2011), no SCC tumors developed in K14-CreER Tet2L/+ p53+/+ or K14-CreER Tet2L/L p53+/+ mice (Figure 3c). SCC tumors developed at low frequency in mice lacking one allele of p53, however, there was no significant difference in tumor penetrance at 500 days between K14-CreER Tet2+/+ p53L/+, K14-CreER Tet2L/+ p53L/+, or K14-CreER Tet2L/L p53L/+ mice, as assessed by Fisher’s Exact test (Table 1). There was also no significant difference between these groups in SCC tumor-free survival assessed by Log-rank test (Figure 3c). In contrast to mice lacking one allele of p53, K14-CreER Tet2L/L p53L/L mice had a significantly shorter SCC-free survival than K14-CreER Tet2L/+ p53L/L mice (p=0.0127, Log-rank test) and K14-CreER Tet2+/+ p53L/L mice (p=0.0308, Log-rank test). K14-CreER Tet2L/L p53L/L mice also had a significantly higher tumor penetrance at 500 days than K14-CreER Tet2+/+ p53L/L mice (83.3% vs. 33.3%, p=0.0106, Fisher’s Exact Test) (Table 1). Finally, while K14-CreER Tet2L/L p53+/+ mice did not develop tumors, SCC lesions formed in K14-CreER Tet2L/L p53L/+ mice at low penetrance at 500 days, which was significantly lower than K14-CreER Tet2L/L p53L/L mice (4.6% vs. 83.3% p<0.0001, Fisher’s Exact Test) (Table 1). In addition, K14-CreER Tet2L/L p53L/L mice had a significantly shorter SCC tumor-free survival than K14-CreER Tet2L/L p53L/+ mice, (p=0.0001, Log-rank test). Tumors developed in various locations, including the back, the legs, and head, and developed from the epidermis. Cutaneous SCC tumors which developed in K14-CreER Tet2L/L p53L/L mice showed keratinization and stained strongly for p63, confirming their squamous identity (Figure 3d). Consistent with our human cSCC data (Figure 1a), we observed low global 5-hmC staining in SCC tumors with loss of Tet2 protein (Figure 3d). These data demonstrate that loss of Tet2 cooperates with loss of Tp53 in stratified epithelial tissues to drive formation of SCC.

Figure 3. Loss of Tet2 cooperates with p53 loss in murine cutaneous SCC tumorigenesis.

Figure 3.

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(a) Immunohistochemical staining of TET2 in normal human skin. Scale bar = 100μm

(b) Immunohistochemical staining of Tet2 (left) and 5-hmC (right) in the skin of mice of indicated genotype following tamoxifen treatment. Scale bar = 50μm

(c) Kaplan-Meyer survival curve of SCC formation in mice of indicated genotype following treatment with 100mg/kg tamoxifen for 5 days. * p<0.05 *** p<0.05 by Multiple measures ANOVA.

(d) H&E and immunohistochemical staining for p63, 5-hmC, and Tet2 in a cSCC tumor from a tamoxifen-treated K14-CreER Tet2L/L p53L/L mouse. Immunohistochemical staining for Tet2 in the normal skin from the same tumor bearing mouse (right). Scale bar = 50μm

5-hmC, 5-hydroxymethylcytosine; cSCC, cutaneous Squamous Cell Carcinoma; SCC, Squamous Cell Carcinoma.

Table 1.

Tumor Penetrance of Spontaneous Squamous Cell Carcinoma.

Genotype # SCC tumor-bearing mice at 500 days # SCC tumor-free mice at 500 days
Tet2+/+, L/+, or L/L p53+/+, L/+, or L/L 0 14
K14-CreER Tet2L/+ p53+/+ 0 15
K14-CreER Tet2L/L p53+/+ 0 16
K14-CreER Tet2+/+ p53L/+ 1 8
K14-CreER Tet2L/+ p53L/+ 0 10
K14-CreER Tet2L/L p53L/+ 1 21
K14-CreER Tet2+/+ p53L/L 6 16
K14-CreER Tet2L/+ p53L/L 0 5
K14-CreER Tet2L/L p53L/L 10 3
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Loss of Tet2 in the epidermis alters patterns of 5-hydroxymethylcytosine

While the regulation of DNA hydroxymethylation has been examined in cultured keratinocytes (Rinaldi et al., 2016), the function of Tet2-dependent regulation of 5-hmC in keratinocytes in vivo is unclear. In order to identify site-specific changes in 5-hmC patterns in the epidermis, K14-CreER Tet2L/L mice were treated with 100mg/kg tamoxifen or vehicle control daily for 5 consecutive days, and then the epidermis was isolated from mice after 2 weeks. Samples from Tet2+/+ or Tet2−/− skin were pooled, DNA was isolated, and genome-wide levels of 5-hmC were assessed by 5-hmC DNA immunoprecipitation followed by sequencing (hMeDIP-Seq). Short-term loss of Tet2 in keratinocytes had minimal effects on global 5-hmC levels, and overall patterns of 5-hmC were similar between Tet2+/+ and Tet2−/− epidermal keratinocytes (Figure 4a). However, analysis of site-specific changes reveled 3615 regions which had significantly increased 5-hmC in Tet2−/− compared to Tet2+/+ mice, while 2039 regions in the genome had significantly reduced 5-hmC (Figure 4b, Supplementary Table S1). Analysis of the location of these differentially hydroxymethylated regions (DMHRs) revealed that the majority were found within gene bodies (Figure 4b). Changes in 5-hmC can alter gene expression through both proximal and long-range effects (Pastor et al., 2013). To link 5-hmC changes to gene expression, we performed RNA-seq on the isolated epidermis of K14-CreER Tet2L/L mice treated with tamoxifen (Tet2−/−) or vehicle control (Tet2+/+) and identified 791 significant genes with greater than 1.5-fold change (Figure 4c, Supplementary Table S2). Gene Ontology analysis (Ashburner et al., 2000) found enrichment in genes related to keratinization, cornification, and Extracellular Matrix organization (Figure 4d). Interestingly, examination of enriched transcription factor binding sites in our gene set using the ChEA database (Lachmann et al., 2010) found enrichment for PcG proteins (Figure 4e), which can interact with DNMT1 to regulate DNA methylation (Vire et al., 2006) and have been found to control lineage selection in the epidermis (Dauber et al., 2016, Lien et al., 2011).

Figure 4. Tet2 regulates an epidermal identity program in the skin.

Figure 4.

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(a) Comparison of merged peak regions from hMeDIP-seq in Tet2+/+ and Tet2−/− epidermis.

(b) Location of differentially hydroxymethylated regions (DHMRs) where 5-hmC is increased in Tet2−/− epidermis (top) or decreased in Tet2−/− epidermis (bottom) compared to Tet2+/+ epidermis.

(c) Volcano plot of transcript levels in Tet2−/− compared to Tet2+/+ epidermis determined by RNA-seq.

(d) Top 5 most significant Gene Ontogeny (GO) terms in RNA-seq data set.

(e) Top 5 most significant ChIP Enrichment Analysis (ChEA) terms in RNA-seq data set.

(f) Overlap between genes upregulated after Tet2 loss in the epidermis and Hair follicle transient amplifying cell (HF-TAC) genes as defined by (Rezza et al., 2016)

(g) Significantly altered genes in the epidermis of mice following Tet2 excision. Bold indicates genes part of the HF-TAC signature.

(h) hMeDIP-seq peaks in Msx1 and Wnt5a genes from murine epidermis of indicated genotype. Boxes highlight significant merged peak regions found in only one sample.

Previous studies have defined gene signatures for various populations in the mouse skin (Rezza et al., 2016) and we compared them to the gene expression changes in the epidermis after loss of Tet2. Interestingly, there was significant overlap between the genes increased after Tet2 excision and hair follicle transient amplifying cell (HF-TACs) genes (Figure 4f), while there was minimal overlap with hair follicle stem cells, outer root sheath cells, hair matrix cells, or interfollicular epidermis cells (Supplementary Figure S1a). Consistent with this, we noted an increase in extracellular matrix genes Lama2, Col15a1, Fbn1, and Spon1 expressed in the hair germ (Tsutsui et al., 2021), as well as key regulators of HF-TACs (Wnt5a, Lef, Msx1) reported to be regulated by PcG proteins (Lien et al., 2011) (Figure 4g). In addition, we noted a reduction in epidermal (Flg, Sprr1b) and sebocyte (Krt7, Scd3) differentiation markers, consistent with the acquisition of a less differentiated HF-TAC cell state. We noted increased transcription and significant increases in 5-hmC levels in the gene body of the Msx1 and Wnt5a genes following Tet2 excision (Figure 4h), consistent with previously reported associations between high 5-hmC levels in genes and transcriptional activation (Ficz et al., 2011). We also observed increased 5-hmC levels at ENCODE identified enhancer regions (Consortium et al., 2020) distal to the Krt7 gene (Supplementary Figure S1b), many of which have been shown to have long-range interactions with the Krt7 gene (Dixon et al., 2012). In total, these data suggest that Tet2 modulates 5-hmC levels to control both proximal and long-range chromatin interactions regulating keratinocyte lineage choice.

DISCUSSION

Reduced global levels of 5-hmC is found in many tumor types, including AML (Konstandin et al., 2011), melanoma (Lian et al., 2012, Saldanha et al., 2017), and Squamous Cell Carcinoma (Cuevas-Nunez et al., 2018, Jawert et al., 2013, Murata et al., 2015) when compared to normal cells. Our data shows that cutaneous SCC, which results in a similar number of deaths per year as melanoma (Karia et al., 2013, Siegel et al., 2021), exhibits globally reduced levels of 5-hmC (Figure 1a, b). Interestingly, unlike melanoma, we did not see a difference between low stage and high stage tumors (Figure 1b), although it should be noted that lower stage tumor numbers were limited (T1, n=10 and T2a n=20). The TET family of enzymes are the main regulators of Cytosine methylation (Ito et al., 2011), leading to great interest in their role in controlling 5-hmC levels in tumors. Mutational inactivation of TET2 was first described in human myeloid cancers (Delhommeau et al., 2009, Jankowska et al., 2009, Tefferi et al., 2009), and all three TET genes have since been shown to be altered in a wide range of tumor types (Jeschke et al., 2016), including Squamous Cell Carcinoma (Figure 1c, d). Previous work in melanoma has shown that the global dysregulation of 5-hmC can be reversed by over-expression of TET2 (Lian et al., 2012), and recent work in murine model systems have demonstrated a tumor-suppressor role for Tet2 in melanoma (Bonvin et al., 2019). We have previously shown reduced levels of 5-hmC in a 4-NQO induced oral carcinogenesis model (Cuevas-Nunez et al., 2018), and it has recently been shown that Tet2 levels are reduced in higher grade 4-NQO induced SCC lesions (Huang et al., 2020), but it was unclear whether Tet2 could in fact act as a tumor suppressor. Here we demonstrate that Tet2 loss can indeed lead to a reduction in 5-hmC levels in epithelial cells (Figures 2a, 3b), and function as a tumor suppressor in carcinogen-induced and spontaneous SCC models (Figures 2d, e, and 3c).

Like many tumor suppressors, inactivation of Tet2 alone is insufficient to cause SCC tumorigenesis, and there were no reported skin phenotypes in germline Tet2−/− mice (Moran-Crusio et al., 2011, Quivoron et al., 2011). Tumor formation in Tet2−/− mice requires a second hit such as Flt3ITD mutation (Shih et al., 2015) or N-RasG12D mutation (Kunimoto et al., 2018) to drive AML, or N-RasQ61K mutation to promote melanoma (Bonvin et al., 2019). Indeed, we find that loss of Tet2 in keratinocytes does not induce SCC tumors, but when combined with loss of Tp53, results in a very high spontaneous tumor incidence and dramatically decreased median survival. Interestingly, Tet2 appears to function as a phenotypic modifier of Tp53 loss in epithelial tissues. Inactivation of Tp53 alone leads to the formation of SCC tumors, but additional loss of Tet2 increases tumor penetrance and reduces tumor-free survival, which contrasts with the lack of phenotype seen with only Tet2 loss (Figure 3c, Table 1). This result is also consistent with human genetic data, where mutation in TP53 is thought to be an early event in SCC formation (Inman et al., 2018, Yilmaz et al., 2017), while TET2 mutations appear to occur at higher frequency in late-stage SCC tumors (Inman et al., 2018, Li et al., 2015).

Consistent with previous results in melanoma (Lian et al., 2012), oral SCC (Cuevas-Nunez et al., 2018), and esophageal SCC (Murata et al., 2015), we have found that global levels of 5-hmC are reduced in cutaneous SCC compared to adjacent skin (Figure 1a, b). Despite these profound changes in tumors, changes in 5-hmC levels in normal skin upon loss of Tet2 were modest. These data could be explained by compensation by Tet1 and Tet3 in the regulation of 5-hmC in the normal epidermis, which may become more dysregulated as cells progress towards malignancy. This compensation model is supported by the fact that some Human SCC tumors have mutations in more than one TET gene (Figure 1c). Alternatively, loss of Tet2 may offer a competitive advantage to cells during tumor formation and a small population of cells in the epidermis with highly reduced 5-hmC may out-compete others. It is worth noting that cSCC tumors do not generally have loss of 5-hmC in all cells, but have heterogenous mix of cells with differing levels of 5-hmC expression. We have found that loss of Tet2 in the epidermis resulted both in site-specific losses and gains in 5-hmC levels (Figure 3). This is consistent with results seen in melanoma (Bonvin et al., 2019), where comparisons between benign nevi and melanoma also showed site-specific gains and losses of 5-hmC. TET2 has been shown not only to convert 5mC to 5hmC, but also to facilitate the subsequent enzymatic reactions converting 5hmC to 5fC and then 5caC (Ito et al., 2010). This suggests that Tet2 may play a functional role both in the conversion of 5-mC to 5-hmC, as well in the subsequent enzymatic reaction to convert 5-hmC to 5-fC in the normal epidermis in vivo. However, it is important to note that these results may be specific to the epidermis, as there is a wide variation in 5-hmC levels across tissue types (Nestor et al., 2012).

TET enzymes are essential for proper lineage commitment and differentiation in embryonic stem cells (Dawlaty et al., 2014, Ficz et al., 2011) and loss of Tet2 has been demonstrated to increase Hematopoietic Stem Cell self-renewal (Moran-Crusio et al., 2011, Quivoron et al., 2011), and alter differentiation of hematopoietic precursors (Ko et al., 2010). In the human epidermis, increased global 5-hmC levels (Lian et al., 2012) and high TET2 expression (Figure 3a) are found in more differentiated layers of the epidermis. We have found that Tet2 loss reduces differentiation genes, while increasing expression of genes associated with HF-TACs (Rezza et al., 2016). Consistent with these findings, K14-CreER Dnmt1−/− mice were reported to have decreased proliferation and reduced upward migration in TA cells in the hair matrix (Li et al., 2012), supporting a role for DNA methylation-dependent regulation of this cell population. Given the links between epidermal lineage choice and regulation by PcG proteins (Dauber et al., 2016, Lien et al., 2011), it is interesting to note the enrichment for PcG-regulated genes that are activated following loss of Tet2 in the epidermis (Figure 4e), as DNMT1, DNMT3A, and DNMT3B, as well as TET1 have been found to interact with PRC2 (Neri et al., 2013, Vire et al., 2006). Tet1, Tet2, and Tet3 have both unique and overlapping roles (Putiri et al., 2014), and important future studies will be needed to dissect the functions of Tet1 and Tet3 in regulating epidermal lineage choice and their interactions with PcG proteins in epidermal lineages.

In total, our data supports the hypothesis that loss of Tet2 in keratinocytes leads to skewing of epidermal lineages, creating an expanded pool of cells that are susceptible to oncogenic transformation. We find that Tet2 loss resulted in a larger number and more advanced lesions following chemical carcinogenesis (Figure 2b). Furthermore, combined loss of Tet2 and Tp53, increased spontaneous SCC tumor development compared to Tp53 loss alone (Figure 3b, Table 1), similar to reports of N-RasQ61K-driven melanomas (Ito et al., 2011). Following exposure to environmental carcinogens this progenitor population may be more likely to progress towards malignant transformation, especially when combined with loss of p53, which compromises repair of DNA damage.

MATERIALS AND METHODS

Experimental Animals

Standard breeding procedures of previously described K14-CreER (JAX stock #005107, (Vasioukhin et al., 1999), p53Flox (JAX stock # 008462, (Marino et al., 2000), and Tet2Flox (JAX stock #017573, (Moran-Crusio et al., 2011) mice were used to generate all cohorts, which were a mixed C57Bl6/J-CD-1 background. In order to excise conditional alleles, mice were injected IP with 100mg/kg tamoxifen (#T5648, Millipore-Sigma) dissolved in Sunflower seed oil (#S5007, Millipore-Sigma) daily for 5 consecutive days. Oral tumors were induced by addition of 100μg/mL 4-Nitroquinoline-N-oxide (#N8141, Millipore-Sigma) to the drinking water, which was changed weekly. After 8 weeks of treatment, mice were given normal drinking water for 16 weeks, and then assessed for tumor numbers (Kanojia and Vaidya, 2006). Tumor grade for 4-NQO lesions were assessed by double blind reading of two pathologists (DW and CGL). For hMeDIP-seq and RNA-seq analysis, K14-CreER Tet2L/L mice were treated with 100mg/kg tamoxifen or vehicle control daily for 5 consecutive days. Two weeks after the first dose of tamoxifen, the epidermis was isolated by soaking samples in 0.2% dispase at 4°C for 1 hour, and then manually separating the epidermis from the dermis.

hMeDIP-seq

K14-CreER Tet2L/L mice were treated with tamoxifen (n=2) or vehicle control (n=2) and samples from each group were pooled. Genomic DNA from murine epithelial preps was purified, sonicated, and then ligated to Illumina barcode adaptors. Ligated DNA was denatured and incubated with a-5hmC antibody (#39769, Active Motif, Carlsbad, CA) overnight at 4°C, and then immunoprecipitated DNA was purified and sequenced using standard Illumina protocols (Xu et al., 2011). 75-nt sequence reads were mapped to the were aligned to the mouse genome (mm10) using the BWA algorithm with default settings and only reads that pass Illumina’s purity filter, align with no more than 2 mismatches, and map uniquely to the genome were used in the subsequent analysis. Differentially hydroxymethylated regions were identified using MACS2 (Zhang et al., 2008).

Tissue microarray

The tissue microarray consisting of cutaneous Squamous Cell Carcinoma specimens and adjacent skin from Mohs surgery performed at Brigham and Women’s Hospital was constructed upon appropriate approval from the institutional review board. Stains were manually scored by a pathologist (CGL) who was blinded to sample identity. Scores were derived by multiplying the percentage of immunoreactive cells (0% = 0; 1–9% = 1; 10–24% = 2; 25–74% = 3; 75–100% = 4) by staining intensity (negative = 1; weak = 2; moderate = 3; strong = 4) as described previously (Lian et al., 2012).

Study Approvals

All animals were housed and treated in accordance with protocols approved by the Brigham and Women’s Hospital Institutional Animal Care and Use Committee (IACUC). The Mass General Brigham IRB approved use of tissues for this study. All human tissue studies used exclusively de-identified and discarded material collected in the course of routine clinical care, for which the Mass General Brigham IRB has determined that signed informed consent was not required.

Supplementary Material

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ACKNOWLEDGEMENTS

We wish to thank Catherine Douds, Ashley Njiru, and Sterline Romain for technical assistance, and Shannan Ho Sui of the Harvard Chan Bioinformatics Core, Harvard T.H. Chan School of Public Health, Boston, MA, for assistance with analysis of hMe-DIP-seq data. Pathology samples were processed by the Dana-Farber/Harvard Cancer Center Specialized Histopathology Core. This research was funded by NIH/NCI grant number CA208298 and the Brigham and Women’s Hospital Department of Dermatology Fund for New Investigators (MRR). RB was funded by a Sun Pharma/SID Innovation Research Fellowship. YW was funded by NIH/NIAMS grant number T32AR007098 (Harvard Dermatology Training Grant).

Abbreviations:

SCC

Squamous Cell Carcinoma

5mC

5-methylcytosine

5-hmC

5-hydroxymethylcytosine

5-fC

5-formylcytosine

5-caC

5-carboxycytosine

TET

ten-eleven translocation

4-NQO

4-Nitroquinoline-N-oxide

Footnotes

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CONFLICT OF INTEREST

C. D. S. is a steering committee member for Castle Biosciences; a steering committee member and consultant for Regeneron Pharmaceuticals; a consultant for Sanofi; has received research funding from Castle Biosciences, Regeneron Pharmaceuticals, Novartis, Genentech, and Merck, and is a chair for NCCN.

All other authors have declared that no conflict of interest exists.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at:

Supplementary Table S1: Merged peak regions from hMeDIP-seq of murine epidermis

Supplementary Table S2: Significantly altered genes in Tet2−/− epidermis from RNA-seq

Supplementary Figure S1: Tet2 regulation of keratinocytes

Data availability statement

The sequence datasets generated and analyzed during this study are available in the National Center for Biotechnology Information Gene Expression Omnibus repository under accession numbers GSE167949 (hMeDIP-seq, RNA-seq).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1750675-supplement-1.docx (16KB, docx)
2
NIHMS1750675-supplement-2.tiff (19.3MB, tiff)
3
NIHMS1750675-supplement-3.xlsx (2.8MB, xlsx)
4
NIHMS1750675-supplement-4.xlsx (29.6MB, xlsx)

Data Availability Statement

The sequence datasets generated and analyzed during this study are available in the National Center for Biotechnology Information Gene Expression Omnibus repository under accession numbers GSE167949 (hMeDIP-seq, RNA-seq).

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