Coordinated activation of DNMT3a and TET2 in cancer stem cell-like cells initiates and sustains drug resistance in hepatocellular carcinoma

doi:10.21203/rs.3.rs-1432174/v1

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Coordinated activation of DNMT3a and TET2 in cancer stem cell-like cells initiates and sustains drug resistance in hepatocellular carcinoma

https://doi.org/10.21203/rs.3.rs-1432174/v1

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Background

Resistance to targeted therapies represents a major hurdle to successfully treat hepatocellular carcinoma (HCC). While epigenetic abnormalities are critical determinants of HCC relapse and therapeutic resistance, the underlying mechanisms are poorly understood. We aimed to address whether and how dysregulated epigenetic regulators have regulatory and functional communications in establishing and maintaining drug resistance.

Methods

HCC resistant cells were characterized by CCK-8, IncuCyte Live-Cell analysis, flow cytometry and wound-healing assays. Target expression was assessed by qPCR and Western blotting. Global and promoter DNA methylation was measured by dotblotting, methylated-DNA immunoprecipitation and enzymatic digestion. Protein interaction, promoter binding and gene mutations of DNMT3a-TET2 was investigated by co-immunoprecipitation, ChIP-qPCR and Sanger sequencing. The regulatory and functional roles of DNMT3a and TET2 were studied by lentivirus infection and puromycin selection. The association of DNMT and TET expression with drug response and survival of HCC patients was assessed by public datasets, spearman correlation coefficients and online tools.

Results

We identified a coordination of DNMT3a and TET2 as an actionable mechanism of drug resistance in HCC. The faster growth and migration of resistant HCC cells were attributed to DNMT3a and TET2 upregulation followed by increased 5mC and 5hmC production. HCC patients with higher DNMT3a and TET2 had a shorter survival time with less responding to sorafenib therapy than those with lower expression. Cancer stem cell-like cells (CSCs) displayed DNMT3a and TET2 overexpression, which were insensitive to sorafenib. Either genetic or pharmacological suppression of DNMT3a or/and TET2 impaired resistant cell growth and oncosphere formation, and restored sorafenib sensitivity. Mechanistically, DNMT3a did not establish a regulatory circuit with TET2, but formed a complex with TET2 and HDAC2. This complex bound the promoters of oncogenes (i.e., CDK1, CCNA2, RASEF), and upregulated them without involving promoter DNA methylation. In contrast, DNMT3a-TET2 crosstalk silences tumor suppressors (i.e., P15, SOCS2) through a corepressor complex with HDAC2 along with increased DNA methylation in their promoters.

Conclusions

We demonstrate that DNMT3a and TET2 act coordinately to regulate HCC cell fate in DNA methylation-dependent and -independent manners, representing strong predictors for drug resistance and poor prognosis, and promising therapeutic targets for refractory HCC.

Hepatocellular carcinoma

sorafenib

epigenetics

drug resistance

DNA methyltransferases

the ten-eleven translocation proteins

DNA methylation

cancer stem cell-like cells

Hepatocellular carcinoma (HCC) represents ~ 90% of all cases of primary liver cancer, and is the second cause of cancer-related death worldwide.¹ Sorafenib is an oral multikinase inhibitor (TKI) targeting several signaling pathways, for instance, the Ras/Raf/MEK/ERK mitogenic signaling, VEGFR and PDGFR etc. When diagnosed, most patients present the metastatic disease, and sorafenib is the only Food and Drug Administration (FDA)-approved first-line systemic therapeutic drug. Despite initial response, the majority of patients display disease progression characterized by acquired sorafenib resistance. As sorafenib inactivates multiple signaling pathways, acquisition of resistance might involve distinct mechanisms. Indeed, several molecular mechanisms have been proposed to explain how HCC cells avoid sorafenib killing, including transport processes,² changes in DNA repair, gene mutations,^3–5 epigenetic aberrations, and dysfunction of compensatory signaling cascades.⁵ However, the precise molecular basis underlying acquired sorafenib resistance still remains barely understood.

DNA methylation involves a covalent chemical modification of DNA. On the one hand, in the presence of S- adenosylmethionine that serves as a methyl donor, a methyl group is added to C-5 position of cytosine residues yielding 5-methylcytosine (5mC). This DNA methylation process is installed by DNA methyltransferases (mainly DNMT1, DNMT3a and DNMT3b). DNMT1 has been proposed to be the maintenance methyltransferase transferring the methylation patterns to daughter cells,⁶ but DNMT3a and DNMT3b perform de novo methylation on unmethylated CpG dinucleotides.⁷ On the other hand, the 5mC can be the substrate of the ten-eleven translocation proteins (TET1, TET2, TET3)-mediated oxidation process leading to 5-hydroxy-methylcytosine (5hmC), whereby promoting locus-specific reversal of DNA methylation.⁸ When dysregulated or/and mutated, DNMTs and TETs break the balance between 5mC and 5hmC production, leading to aggressive cancer growth.^9,10 Notably, loss-of-function TET2 mutations are frequent in leukemia,^11,12 but much low mutation rate has been observed in HCC (< 5%).¹³ Although all three TETs are considered to be tumor suppressors in various cancers, evidence is emerging that TET1 and TET2 upregulation has oncogenic potential in certain cancer types, like HCC,¹⁴ leukemia¹⁵ and breast cancer.¹⁶ Mechanistically, aberrant epigenetic changes have been appreciated in HCC tumorigenesis¹⁷ and drug resistance in other cancers,^9,18 but the underlying mechanisms have been poorly defined so far. Moreover, DNMTs and TETs are scaffold proteins that recruit other transcription factors to regulate target expression, but it is largely unknown whether and how such regulatory activities promote resistance to sorafenib in HCC.

In this study, we explored the role of DNMT3a in coordination with TET2 in promoting HCC cell resistance to sorafenib, as well as the mechanism by which DNMT3a and TET2 regulate resistance-sustaining genes. We demonstrate that a functional cooperation of DNMT3a and TET2 is required for HCC cells to escape sorafenib killing. Mechanistically, upregulation of DNMT3a and TET2 further silences TSGs through promoter DNA hypermethylation; In parallel, TET2 did not have a regulatory feedback loop with DNMT3a, but forms a complex with DNMT3a and HDAC2. This complex binds oncogene promoters followed by oncogenic upregulation in resistant cells. Either genetic or pharmacological inactivation of DNMT3a or/and TET2 impairs sorafenib resistant cell growth. Our discoveries demonstrate DNMT3a and TET2 coordination as a robust target, whose disruption represents a promising strategy to increase the therapeutic efficacy of sorafenib in patients with refractory HCC.

Cell lines, cell culture, vectors and reagents

The Hep3b and Huh7 cell lines were obtained from American Type Culture Collection with no further authentication or testing for mycoplasma. The cell lines were grown in MEM (Hep3b, CORNING #10-010-CV) or DMEM (Huh7, CORNING #10-013-CV), supplemented with 10% FBS (Gibco by Life Technologies^TM, #16140-071) and Antibiotic-Antimycotic (Gibco by Life Technologies^TM, #15240062) at 37°C under 5% CO₂. No cell line used in this paper is listed in the database of commonly misidentified cell lines maintained by ICLAC (International Cell Line Authentication Committee). The shRNAs against DNMT3a (V2LHS_202509, V2LHS_74666, V2LHS_74668), TET2 (V2LHS_227656, V2LHS_266084) and the negative control (pGIPZ) vectors were obtained from BMGC RNAi (University of Minnesota). TET2 inhibitor Bobcat339 (#408006) was obtained from MedKoo Biosciences, but Sorafenib (#SML2653) from Sigma-Aldrich. Solutions of Bobcat339 were prepared in dimethyl sulfoxide (DMSO) at 100 mM, while Solutions of Sorafenib were prepared in DMSO at 10 mM.

In vitro adaption of sorafenib resistant cells

Hep3b and Huh7 cell lines were passaged with low concentration of sorafenib (0.1 µM) and sequentially cultured in increasing concentrations of sorafenib (0.3, 1, 2 µM) for 8 weeks. Cells cultured in parallel without drugs (DMSO) served as parental negative controls. Cells were considered resistant when they could routinely grow in respective medium containing 2 µM sorafenib.

Lentivirus production and infection for gene knockdown

For virus production, about 3.8 x 10⁶ HEK-293 cells were plated in a 10 cm cell culture dish. After 24h, cells were transfected with 6 µg of target or scrambled control plasmids using calcium phosphate transfection reagent (CalPhos™ Mammalian Transfection Kit). The lentiviruses were harvested at 48 and 72 hours after transfection and concentrated according to the protocol of the Lenti-X™ Concentrator (#631232, Clotech). For virus infection, about 1 x 10⁶HCC cells were infected by the lentiviruses using Polybrene (final concentrate 4 ug/ml) in 1ml respective medium and Puromycin (final concentration 2 µg/ml) was added to select the stable transformants 24 hours post-infection.

Cell proliferation assays

Hep3b or Huh7 cells with various treatments were seeded into a 24-well or 12-well culture plates for 24 hours. Then the plates were incubated in the IncuCyte S3 Live-Cell Analysis System for real-time analysis. Data were analyzed by the software provided. The total area of phase or green fluorescent cells for each picture was calculated, which represents the confluence of Hep3b or Huh7 cells.

Wound healing assays

Cells with various treatments were first seeded into a 96-well culture plates, and on the 2nd day, subjected to the wound healing assays. The plates were then put into the IncuCyte S3 Live-Cell Analysis System for real-time analysis. Cell migration toward the wound was photographed under IncucyteS3, and the migration distance was assessed by CorelDRAWX5 Software.

Apoptosis and cell cycle assays

Cell apoptosis and cell cycle assays were performed using Annexin V-PI Apoptosis Detection Kit I (#556547, BD Pharmingen^TM) and CycleTEST PLUS DNA Reagent Kit (#340242, BD Pharmingen^TM), respectively, according to the manufacturer’s instruction, and followed by flow cytometry analysis.

CCK-8 assays

CCK-8 assays were performed using Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, #CK04-11). Briefly, the parental and resistant Hep3b or Huh7 cells (1.5 × 10⁴) in MEM or DMEM medium (100 µl) were dispensed into 96-well flat bottomed microplates and drugs were added after 24 hours incubation. The cells were cultured for additional 24 or 48 hours, and CCK-8 reagent (10 µl) was added to each well. Then the microplates were incubated at 37 °C for another 1~2 hours. Absorbance was measured at 450 nm using a microplate reader and the results were expressed as a ratio of the treated over untreated cells (as 100%). Four wells were sampled per each experimental group in a given experiment. Averages are reported ± SD.

Oncosphere-forming assays

About 1,000 Hep3b or Huh7 cells were seeded in appropriate medium in each well of a Corning® Costar® Ultra-Low attachment plate (#3473), and incubated for about 7 days. During the incubation, the cells were pipetted every 2 days. Then the number of oncospheres were counted and images were taken.

DNA Dotblotting

Genomic DNA was extracted using DNA Blood/Tissue Kit (QIAGEN), denatured and subjected to dotblotting analysis using antibodies against 5mC or 5hmC (Supplementary table 2) as previously described.¹⁹^,²⁰ The DNA spotted membrane was stained with 0.02% methylene blue (Sigma) in 0.5 M sodium acetate (pH 5.0) for DNA loading control.

Promoter methylation assays

Genomic DNA was isolated from parental and sorafenib^R HCC cells or sorafenib^R Hep3b cells with TET2 or DNMT3a gene knockdown or treatment with Bobcat339. Equal amount of DNA (1 μg) from these cells was digested by HpaII or BstUI for 2-4 hours. The digested DNA was cleaned by PCR purification kit (QIAGEN) and subjected to SYBR Green PCR using primers specific for TSG promoters (CpG enriched region; Supplementary table 1).

Co-immunoprecipitation (Co-IP) and Western blotting

The whole cellular lysates were prepared by harvesting the cells in 1 × cell lysis buffer (20 mM HEPES (pH 7.0), 150 mM NaCl and 0.1% NP40) supplemented with 1 mM phenylmethane sulfonyl fluoride (PMSF, #10837091001, Sigma), 1 × Phosphatase Inhibitor Cocktail 2 and 3 (Sigma, #P5726, P0044), and 1 × protease inhibitors (protease inhibitor cocktail set III, Calbiochem-Novabiochem, #539134). Approximately 0.5-1 mg total protein lysate was precleared with 70 μL of slurry of protein G Dynabeads (Thermo Fisher, #10004D) for 2 hours at 4 °C. Dynabeads (70 μL) were coated with 2 to 5 μg respective antibodies at 4 °C overnight. The dynabeads were washed in 1 × cell lysis buffer and boiled in 1 × loading buffer for 5-10 min to elute proteins. The immunoprecipitates and whole cell lysates were subjected to Western blotting using our established methods.¹⁹^,²¹^,²² The Western blots were quantified using the Image J Software from the U.S. National Institutes of Health. The antibodies used are listed in supplementary table 2.

Chromatin Immunoprecipitation (ChIP)

ChIP assays were performed using the Chromatin Immunoprecipitation Assay Kit (Millipore Sigma, #17-295) according to the manufacturer’s standard protocol. DNA was quantified using qRT-PCR with SYBR green incorporation (Applied Biosystems). The antibodies used are listed in Supplementary Table 2. The primers for gene expression are listed in supplementary table 1.

RNA extraction, cDNA preparation, quantitative PCR (qPCR) and sanger sequencing

RNA was extracted using the miRNeasy Kit (QIAGEN) and reverse transcription for cDNA was performed using the High-Capacity cDNA Reverse Transcription Kit (#4368814, Applied Biosystems). The SYBR-Green qPCR (#4309155, Applied Biosystems) was used to measure gene expression. The 18S levels were used for normalization and target expression was analyzed using the DCT approach. Regarding sanger sequencing for gene mutations, the cDNA for genes including DNMT3a and TET2 were synthesized and subjected to sequencing. The primers are lists in Supplementary Table S1.

Methylated DNA immunoprecipitation (MeDIP) assays

MeDIP assays were performed using the MeDIP Assay Kit (#55009, Active Motif) according to the manufacturer’s standard protocol. Briefly, genomic DNA was extracted by DNA Blood/Tissue Kit, and 10-20 ug DNA was sonicated to obtain fragments ranging 200–500 bp. The sonicated DNA fragments were mixed with 5-mC antibody and magnetic beads in IP incubation mix for overnight at 4 °C on a rotating wheel. Beads were then washed with wash buffer 1 for three times and once with 100 μL of ice-cold wash buffer 2. The supernatant was discarded, and bead pellets were preserved for DNA elution. Two IP and two input samples were immunoprecipitated per MeDIP array. qPCR was used to quantify the enriched DNA for both Input and IP samples. MeDIP efficiency using qPCR was calculated using following formula: %(meDNA-IP/Total input) = 2^[(Ct(10%input)-3.32) – Ct(meDNA-IP)] x 100%.

Analysis of gene expression omnibus (GEO) data analysis

Five expression microarray series, GDS4887, GSE45267, GSE54236, GSE109211, GSE63838 containing HCC tumor and nontumor samples were downloaded from the Gene Expression Omnibus (GEO) datasets, and analyzed for the expression of DNMTs and TETs. These samples were normalized, managed and analyzed by GraphPad Prism 5 Software using Spearman correlation coefficients and online tools (protein atlas, Gepia2).

Statistical analysis

The statistical analysis was performed using the Student’s t test. All analyses were performed using the GraphPad Prism 5 Software. p < 0.05 was considered statistically significant. All p values were two-tailed. All criteria were pre-established. No statistical method was used to predetermine sample size and the sample size for all experiments was not chosen with consideration of adequate power to detect a pre-specified effect size. In vitro experiments, such as qPCR, Western blotting, dotblotting, cell proliferation and apoptosis assays, wound-healing assays were routinely repeated three times unless indicated otherwise in Figure legends or main text. For each Figure, the statistical tests were justified as appropriate.

Sorafenib^R HCC cells display improved viability, enhanced migration and reduced apoptosis after transient sorafenib treatment

To gain insights into the mechanisms of sorafenib resistance, we initially modeled the response of HCC Hep3b and Huh7 cells to sorafenib by continuously culturing them with a step-wise increase of drug dosages for 8 weeks. The final concentration of sorafenib was 2 µM, which exerted sufficient inhibitory action and was in the range of clinically achievable levels.²³^,²⁴ To characterize these sorafenib-selected cells, we first measured the proliferation of parental and resistant (sorafenib^R) cells upon transient exposure to 2 µM sorafenib. Although all parental controls displayed significant decreases of cell proliferation, the sorafenib^R cells did not show obvious difference in cell proliferate (Fig. 1a and Fig. S1a), upon exposure to sorafenib. This was further confirmed by the enhanced wound-healing capability in sorafenib^R versus parental Hep3b and Huh7 cells (Fig. 1b and Fig. S1b). Second, flow cytometry analysis revealed that transient sorafenib treatment does not have obvious alterations in cell apoptosis and cell viability in sorafenib^R Hep3b and Huh7 cells, but significantly promotes cell apoptosis and impairs cell viability in parental controls (Fig. 1c and 1d). We did not see obvious changes in cell cycles in both parental and sorafenib^R Hep3b and Huh7 cells when exposed to 2 µM sorafenib (Fig. S1c). Third, we observed that, as compared with parental cells, the oncosphere formation is enhanced in sorafenib^R with less affected by sorafenib treatment (Fig. 1e; Fig. S1d), and resistant cells exhibit a higher rate of cell proliferation in drug free medium (Fig. 1f; Fig. S1e). These findings are in line with our previous data showing that lung cancer cells resistant to tyrosine kinase inhibitors (i.e., midostaurin) migrate and grow at a faster pace than parental cells in vitro, and possess higher tumorigenic potential in vivo.⁹

As sorafenib discontinuation is a frequent event in HCC patients, we mimicked sorafenib “holiday” by culturing sorafenib^R cells in drug-free medium for 14 days (released cells). Interestingly, the released cells displayed proliferation impairment, which is supported by a reduction of cell proliferation, and increase of cell apoptosis in the presence of sorafenib, when compared with parental and sorafenib^R cells (Fig. 1g; Fig. S1f). These results indicate that partial re-acquisition of sensitivity to sorafenib leads to a transient proliferation arrest upon drug withdrawal, phenocopying the drug holiday effect seen in the clinic. This is consistent with our recent findings in leukemia that resistance to TKIs (i.e., nilotinib, imatinib) displays reversible features or a transient proliferation arrest upon exposure to the same drug.²¹ Finally, we performed sequencing for epigenetic regulators, for example, DNMT3a and TET2 that are frequently mutated in cancers, in sorafenib^R Hep3b and Huh7 cells. We did not find any acquired mutations in these genes. Taken together, these results suggest that non-genetic mechanisms could be essential in sorafenib resistance, and although generated in vitro, our drug-resistant HCC cell line derivatives faithfully recapitulate clinical drug resistance.

HCC cells with sorafenib-acquired resistance have activation of pathways involved in DNA methylation

To identify critical molecules that promote sorafenib resistance, first we examined the expression of DNA methyltransferases (DNMTs) in parental and sorafenib^R Hep3b and Huh7 cells, because epigenetic aberration (i.e., DNA hypermethylation) is becoming increasingly important in the development of TKI resistance.⁹^,²¹ The results from qPCR and Western blotting disclosed that expression of DNMT3a and DNMT3b, two de novo DNA methyltransferases,⁷ is increased at both RNA and protein levels, but expression of DNMT1, a maintenance DNMT,²⁵ as well as HDAC2 and HDAC3, histone protein modifiers, is not obviously changed (Fig. 2a and 2b). As DNA methylation is installed by DNMTs via adding a methyl group to C-5 position of cytosine residues yielding 5-methylcytosine (5mC), we proceeded to determine whether 5mC amount is changed in sorafenib^R cells. The DNA dotblotting by anti-5mC antibody¹⁰^,²⁶ revealed that 5mC production is significantly elevated in sorafenib^R Hep3b and Huh7 cells compared with parental counterparts (Fig. 2c and 2d). These results extend previous findings showing that 5mC amount is markedly increased in leukemia cells resistant to nilotinib¹⁰ and in lung cancer cells resistant to midostaurin.⁹

It has been shown that the TET methylcytosine dioxygenases catalyze the conversion of 5mC to 5hmC, resulting in DNA demethylation.⁸^,^27-29 To further understand the role of DNA methylation in sorafenib resistance, we performed qPCR and Western blot for changes of TETs, and found that expression of TET1, TET2 and TET3 is significantly upregulated at both RNA and protein levels in sorafenib^R compared with parental cells (Fig. 2e and 2f). Dotblotting analysis using anti-5hmC antibody revealed that 5hmC level is much higher in sorafenib^R Hep3b and Huh7 cells than that in parental counterparts (Fig. 2g and 2h), in agreement with the methylcytosine dioxygenase activities of TET1, TET2 and TET3. Collectively, these findings suggest that long-term exposure to sorafenib alters the functions of DNA methylation machinery in HCC cells.

Knockdown of DNMT3a and TET2 impairs sorafenib^R HCC cell growth

Having demonstrated the upregulation of DNMT3a, a de novo DNA methyltransferase,⁷ in sorafenib^R HCC cells, next we sought to determine the biological functions of DNMT3a aberrations. Sorafenib^R Hep3b and Huh7 cells were infected with DNMT3a shRNA or scrambled vectors for 24 hours and further selected by 2 µM puromycin for additional 96 hours. First, the efficacy of virus infection was verified by the high rate of GFP/fluorescent cells (Fig. S2a). The shRNA-3, which showed the most DNMT3a reduction, was used for further investigations. As shown in Fig. 3a and 3b, both RNA and protein expression of DNMT3a was markedly decreased in DNMT3a shRNA compared with control group. The specificity of DNMT3a knockdown was supported by the unchanged expression of DNMT1, DNMT3b and TET2 in DNMT3a shRNA versus control cells. Second, the results from dotblotting showed that 5mC production is much lower in DNMT3a-depleted cells than that in control groups (Fig. 3c and 3d). Interestingly, DNMT3a knockdown led to a decrease of 5hmC abundance even when TET2 protein levels are not changed. This may result from the reduction of 5mC amount, the original substrate of TET2. Third, these DNMT3a depleted cells were subjected to proliferation and wound-healing assays. We observed that cells with DNMT3a knockdown could proliferate and migrate at a much lower rate, in a time-dependent manner, than that in control counterparts (Fig. 3e and 3f; Fig. S2a and Fig. S2b). These findings support an important contribution of DNMT3a to sorafenib^R cell growth.

Having shown that TET2 is upregulated in sorafenib^R compared with parental cells, next we sought to examine whether TET2 expression is essential for sorafenib^R cell growth. To this end, sorafenib^R Hep3b and Huh7 cells were infected with TET2 shRNA or control vectors for 24 hours followed by puromycin selection for additional 96 hours. The high rate of GFP/fluorescent cells indicated the high efficacy of virus infection (Fig. S2c). The results from qPCR and Western blot demonstrated the efficient knockdown of TET2 gene, and the specificity of TET2 knockdown was supported by the unchanged levels of TET1, TET3 and DNMT3a in TET2 shRNA-transfected cells (Fig. 3g and 3h). As TET2 is a methylcytosine dioxygenase, we speculated that TET2 ablation may decrease 5hmC production. Indeed, the results from dotblotting using 5hmC antibody support that sorafenib^R Hep3b and Huh7 cells have much lower levels of 5hmC than parental cells (Fig. 3i and 3j). Notably, TET2 knockdown led to an increase of 5mC abundance even when DNMT3a protein expression are not changed. This may be attributed to the downregulation of TET2, leading to less conversion of 5mC to 5hmC.

To investigate the biological outcomes of TET2 knockdown, the sorafenib^R Hep3b and Huh7 cells with TET2 depletion were subjected to proliferation and wound-healing assays. We found that, compared to those with scrambled controls, the cells with TET2 knockdown proliferate at a much slower rate (Fig. 3k, Fig. S2c) and migrate at a much shorter distance (Fig. 3l, Fig. S2d), which occurs in a time-dependent manner. These findings suggest that TET2 is required for the aggressive proliferation and migration of sorafenib^R HCC cells.

CSCs with upregulation of DNMT3a and TET2 are more tolerant to sorafenib-induced cell death

Given the highly heterogeneous of HCC cells and the role of oncospheres in developing drug resistance, we performed oncosphere-forming assays in HCC Hep3b and Huh7 cells, and found that about 6% and 5% cells (ratio: Onco/total) can form oncospheres, respectively (Fig. 4a). To examine the drug sensitivity of oncospheres, we replated oncospheres and treated them with different doses of sorafenib. As shown in Fig. 4b, the replated oncosphere cells displayed IC₅₀ values to sorafenib much larger than those exhibited by their parental counterparts, suggesting that oncospheres may mediate, at least partially, the development and maintenance of sorafenib resistance. In line with this, sorafenib^R Hep3b and Huh7 cells had a greater tendency to form oncospheres than parental cells (Fig. 4c). To identify essential regulators for oncosphere formation, we examined the levels of DNMTs and TETs. We found that expression of DNMT3a and TET2 at RNA and protein levels is highly elevated in oncosphere compared to parental cells (Fig. 4d and 4e). To assess the involvement of DNMT3a and TET2 in oncosphere formation, we used shRNA lentiviruses to knock down them, and found that DNMT3a or TET2 knockdown inhibits the oncosphere growth supported by reduction of oncosphere number without obvious changes of oncosphere sizes (Fig. 4f). Further, we found that oncosphere cells express elevated levels of stem cell makers like CD133, CD25, CD44 and c-KIT (Fig. 4g). Therefore, we propose that upregulation of DNMT3a and TET2 in CSCs is essential for the development of sorafenib resistance.

Dysregulation of DNMTs and TETs is linked to sorafenib responses and survivals in HCC patients

To explore the clinical implications of DNMT upregulation in sorafenib^R HCC cells, we sought to determine if DNMT3a expression can predict HCC patient responses receiving sorafenib therapy. We first analyzed DNMT3a mRNA levels in public datasets GDS4887, GSE45267 GSE54236 and GSE109211, and found that DNMT3a is significantly upregulated in HCC tumors compared to non-tumor tissues (Fig. 5a). Importantly, sorafenib non-responders (n = 21) had higher levels of DNMT3a expression than responders (n = 40) (Fig. 5b). Notably, we also found that DNMT1 and DNMT3b expression is higher in HCC tumors than non-tumor tissues as well as in sorafenib non-responders than responders (Fig. S3a). To validate prognostic implications for DNMT3a overexpression, we analyzed the survival of HCC patients from GSE54236, GSE109211 or used online tools (protein atlas, Gepia2) to examine the association between patient outcomes and DNMT3a levels. We observed that HCC patients with higher DNMT3a expression have significantly shorter survival time than patients with lower levels (Fig. 5c and 5d). Importantly, sorafenib non-responding patients, who have higher DNMT3a expression, had a shorter survival time than responders carrying lower DNMT3a expression (Fig. 5e). We also assessed the association of patient outcomes with the expression of DNMT1 and DNMT3b, and found that upregulation of DNMT1 and DNMT3b corresponds with short overall survival in HCC patients as well as in sorafenib non-responders (Fig. S3b and 3c). Given that DNMT3a is a de novo DNA methyltransferase, these findings suggest that DNMT3a upregulation followed by 5mC increase is more essential in sustaining sorafenib resistance.

To demonstrate the clinical significance of TET2 upregulation in HCC patients, we analyzed public datasets GSE45267, GSE54236 and GSE109211 for TET2 mRNA levels. We found that, in general, TET2 expression has a trend toward upregulation in HCC tumors compared to normal tissues (Fig. 5f), which is required to be validated in a larger cohort of patients. Importantly, sorafenib nonresponders (n = 21) had significantly higher levels of TET2 (p < 0.05) than responders (n = 40) (Fig. 5g). Notably, we also found that TET1 is higher in HCC tumors than non-tumor tissues and in sorafenib non-responders than responders (Fig. S3d). To explore the prognostic implications of TET2 overexpression, we analyzed the survival of HCC patients from GSE54236, GSE109211 or used online tools (protein atlas, Gepia2) to examine the association between patient outcomes and TET2 levels. We observed that HCC patients with higher TET2 have significantly shorter survival time than those with lower TET2 (Fig. 5h). Importantly, sorafenib non-responding patients, who have higher TET2 expression, had a small survival probability than responders carrying lower TET2 expression (Fig. 5i). We also assessed the association of patient outcomes with the expression of TET1 and TET3, and found that upregulation of TET1 and TET3 corresponds with short overall survival in HCC patients (Fig. S3e and S3f). These findings extended the previous studies showing that TET2 is upregulated in HCC cells and may be important for HCC cell growth.¹⁴ Collectively, our data suggest that TET2 is an essential regulator of HCC sorafenibresistance.

Pharmacological targeting of TET2 impairs sorafenib^Rcell growth

The findings, which TET2 is upregulated in sorafenib^R cells, and TET2 gene knockdown inhibits resistant cell growth, imply that TET2 could be a pharmacological target. To test this, we treated sorafenib^R Hep3b and Huh7 cells with bobcat339, a selective cytosine-based TET enzyme inhibitor.³⁰ Western blotting revealed that exposure to bobcat339 does not change protein expression of either TET2 nor TET1 and TET3 (Fig. 6a), and dotblotting analysis showed that bobcat339 treatment reduces DNA 5-hmC abundance (Fig.6b and 6c) without obvious changes of 5mC amount (Fig.6d and 6e), in line with the concept that bobcat339 does not inhibit de novo methyltransferase DNMT3a.³⁰

To assess if exposure to bobcat339 inhibits sorafenib^R cell growth, we performed flow cytometry and observed that cell apoptosis is significantly increased in the presence versus absence of bobcat339 (Fig. 6f and 6g). Further, proliferation and wound-healing assays found that sorafenib^R cells treated with bobcat339 proliferate and migrate at a much lower rate than untreated cells (Fig. 6h and 6i; Fig. S4a and Fig. S4b). Furthermore, combination of sorafenib with bobcat339 resulted in more pronounced inhibition of sorafenib^R cell proliferation than the treatment with single reagents (Fig. 6j; Fig. S4c). Collectively, these results support that TET2 inhibitors may be promising therapeutic reagents in overcoming sorafenib resistance, which merits thorough investigations in vivo.

DNMT3a and TET2 regulate sorafenib sensitivity and coordinately modulate sorafenib^Rcell proliferation

Given that both DNMT3a and TET2 are upregulated in resistant cells, and as knockdown of DNMT3a or TET2 suppresses resistant cell growth, we speculated that DNMT3a or TET2 expression may be associated with sorafenib sensitivity. To this end, DNMT3a or TET2 was knocked down in sorafenib^R Hep3b and Huh7 cells having their overexpression. These cells were treated with 2 µM of sorafenib for indicated time points. Cell proliferation assays revealed that depletion of DNMT3a or TET2 sensitizes sorafenib^R cells to sorafenib-inhibited cell proliferation (Fig. 7a and 7b), and wound-healing studies uncovered that DNMT3a or TET2 knockdown enhances sorafenib-impaired cell migration (Fig. 7c and 7d). These findings suggest that DNMT3a or TET2 expression is required to maintain sorafenib-resistant phenotypes of HCC cells.

To establish the role of DNMT3a and TET2 interaction on sorafenib^R cell growth, we knocked down either DNMT3a and TET2 alone or both. The cell proliferation assays revealed that concurrent knockdown of DNMT3a and TET2 leads to more robust inhibition of cell proliferation and migration than that of a single gene change (Fig. 7e and 7f), suggesting that DNMT3a and TET2 have functional cooperation in sustaining proliferation and migration of sorafenib^R HCC cells.

DNMT3a physically interacts with TET2, but does not form a regulatory loop, to enhance sorafenib^R resistant phenotypes

Because DNMT3a and TET2 are upregulated simultaneously in sorafenib^R HCC cells, we first determined if DNMT3a regulates TET2 transcription or vice versa. By using online tools (GEPIA 2) and analyzing public data GDS4887, GSE45267 and GSE54236, we did not find an obvious correlation between DNMT3a and TET2 (Fig. S5a and S5b). These findings, together with the observations that DNMT3a knockdown does not change TET2 protein expression or vice versa (Ref. Fig. 3b and 3h), suggest that DNMT3a and TET2 don’t transcriptionally regulate each other. We then examine if DNMT3a and TET2 have protein interactions in HCC cells. Co-IP assays revealed that DNMT3a forms a complex with TET2 and HDAC2 in both sorafenib^R and parental HCC cells with a slight increase of their interaction potential in sorafenib^R cells (Fig. S5c and S5d). The protein interaction among DNMT3a, TET2 and HDAC2 was further confirmed by the results from string-interaction assays (Fig. S5e; https://string-db.org), which was consistent with previous studies showing that HDAC2 and TET2 are co-transcription factors of DNMT3a.^31-33 Given the dual function of TET2 in cancers,³³ these findings suggest that a transcriptional complex of DNMT3a, TET2 and HDAC2 exists in sorafenib^R HCC cells.

Tumor suppressor genes are further silenced in sorafenib^R HCC cells by DNMT3a- mediated promoter DNA hypermethylation

To define the mechanisms that DNA methylation aberrations contribute to faster growth of sorafenib^R cells, we examined the changes of epigenetically silenced tumor suppressor genes (TSGs), for example, p15, p16, p18, FHIT, SOCS1 and SOCS2, which are frequently silenced by promoter DNA methylation and their downregulation predicts worse prognosis in HCC patients. The results from qPCR showed that expression of p15 and SOCS2 is largely decreased in sorafenib^R compared with parental Hep3b and Huh7 cells, but no obvious changes were detected in the expression of p16, p18, FHIT, and SOCS1 (Fig. 8a). These results suggest that further TSG silencing is required for the survival and faster proliferation of sorafenib^R cells.

As epigenetic regulators TET2 and DNMT3a are upregulated in sorafenib^R cells, we reasoned that overexpression of TET2 and DNMT3a may account for TSG silencing. To test this, we knocked down DNMT3a or disrupted TET2 activity via bobcat339 in sorafenib^R Hep3b and Huh7 cells, and assessed expression of these TSGs. As expected, DNMT3a ablation significantly increased the levels of p15 and SOCS2, but genetic or pharmacological inactivation of TET2 did not obviously change their expression. Notably, co-knockdown of TET2 and DNMT3a led to more pronounced upregulation of p15 and SOCS2 (Fig. 8b), suggesting that TET2 and DNMT3a coordinately silence TSGs in sorafenib^R cells.

To investigate the methylation status of p15 and SOCS2 promoters, genomic DNA from sorafenib^R and parental cells was digested with the restriction enzyme HpaII cutting only nonmethylated sites or BstUI cutting only methylated sites. Decitabine-treated cells were used as positive controls. Following digestion, DNA was analyzed by qPCR using primers specific to p15 or SOCS2 gene promoter containing CpG islands. Efficient digestion by HpaII or BstuI leads to stronger amplification in hypomethylated or hypermethylated p15 and SOCS2 promoters, respectively, in sorafenib^R cells compared to parental controls. Consistent with the silencing data (see Fig. 8a) and opposite to decitabine-treated controls, these data provide the evidence that the p15 and SOCS2 promoters are more hypermethylated in sorafenib^R cells than that in parental controls (Fig. 8c). To precisely measure the alterations of promoter methylation, we performed MeDIP assays, in which methylated DNA was enriched by 5mC antibody and quantified by qPCR. Consistent with the findings from enzyme digestion (see Fig. 8c), the levels of 5mC in the promoters of p15 and SOCS2 were significantly increased in sorafenib^R cells compared to parental controls (Fig. 8d). To address how TSG promoters become hypermethylated, we performed ChIP assays, and found that both DNMT3a and TET2 bind the promoters of p15 and SOCS2 (Fig. 8e), which are in line with the physical protein interactions between DNMT3a and TET2, leading to DNA hypermethylation that interacts with HDAC2 in the promoter to further silence TSGs.

DNMT3a and TET2 activate cell proliferation genes through promoter binding in a DNA methylation-independent manner

To further understand how sorafenib^R cells more aggressively proliferate and migrate, we first examined the expression of certain oncogenes, like CDK1, CCNA2 and RASEF. We found that these oncogenes are highly elevated in sorafenibR compared to parental cells (Fig. 8f). These results suggest that, in addition to TSG silencing, oncogenic upregulation may contribute to the survival and faster proliferation of sorafenib^R cells. Second, inactivation of either TET2 or DNMT3a led to a decrease of oncogene expression, but we did not see a synergistic effect on oncogenic downregulation upon knockdown of both TET2 and DNMT3a (Fig. 8g). Third, the assays of enzymatic digestion from bulk DNA did not see obvious changes of DNA methylation in the promoters of oncogenes CDK1, CCNA2 and RASEF (Fig. S6a). Instead, MeDIP assays revealed a slight decrease of 5mC in the promoters of CDK1 and CCNA2, but not RASEF (Fig. S6b). Fourth, ChIP analysis disclosed that DNMT3a, TET2 and HDAC2 are enriched in the promoters of oncogenes CDK1, CCNA2 and RASEF (Fig. S6c), although such binding did not increase the levels of DNA methylation in sorafenib^R cells. These findings strongly support that both DNMT3a and TET2 have DNA methylation-independent functions.³⁴^,³⁵

Sorafenib remains one of the limited treatment options and has become the standard therapy for advanced HCC patients. However, clinical responses to sorafenib are often short-lived, and many patients acquire drug resistance and experience disease recurrence. Such disappointing outcomes suggest that successful treatment of advanced HCC patients would require discovery of new molecular rules of acquired sorafenib resistance and, on that basis, implementation of effective treatment regimens. In this study, we present evidence, for the first time, showing that 1) in resistant cells, DNMT3a and TET2 are upregulated, the global and gene-specific DNA methylation is increased, and TSGs are further silenced, but cell proliferation genes are activated. CSCs with overexpression of DNMT3a and TET2 have higher potential to form oncosphere and less sorafenib sensitivity; 2) clinically, upregulation of DNMT3a and TET2 predicts less drug responses and worse prognosis in sorafenib-treated HCC patients; 3) mechanistically, we proved that DNMT3a-TET2 complex binds target promoters and coordinately regulate TSGs and cell proliferation genes via DNA methylation-dependent or -independent manners (Fig. S7). Our work uncovers a critical DNMT3a-TET2 coordination that acts to drive HCC resistance to sorafenib, thus shedding a new light on the molecular biology of cancer drug resistance. Our findings suggest that coordinated silencing of DNMT3a and TET2 is an effective approach to treat refractory or relapsed HCC patients.

Given that sorafenib targets different signaling pathways, the molecular pathways underlying acquired resistance could be multiple. We focused on aberrant epigenetics/DNA methylation, because 1) genetic mutations fail to explain why TKI (i.e., sorafenib) resistant phenotypes are partially reversible;^9,21,36 2) under TKI selection, epigenetic changes accumulate as cell population evolves and diversifies³⁷ at higher rates than genetic alterations. In addition, changes in resistant phenotypes and epigenetic modifications share features: dynamic and reversible;^38–43 3) we and others showed that aberrant epigenetics (i.e., DNA methylation) significantly contributes to TKI resistance;^9,21,39 4) abnormal DNA methylation indeed has been appreciated in sorafenib resistance, but the regulatory and functional roles of DNA methylation regulators are still barely defined. As HCC is highly heterogeneous within the tumors, we speculated that, upon exposure to sorafenib, the dynamic and reversible traits of DNA methylation allow TSGs to be rapidly and further epigenetically silenced or oncogenes to be activated, thus helping a subpopulation of cells survive and proliferate through sorafenib killing. Indeed, our studies identified a previously unknown molecular rule, a DNMT3a-TET2 crosstalk, in determining HCC cell fate when facing sorafenib-imposed selective pressure. First, CSCs have DNMT3a and TET2 upregulation, higher potential to form oncosphere, and are more tolerant to sorafenib. These CSCs could initially survive and proliferate leading to resistant population. Second, the levels of DNMT3a, TET2 and DNA methylation are highly elevated in resistant vs. parental cells. As a consequence, TSGs (i.e., P15, SOCS2) are further silenced and oncogenes (i.e., CDK1, CCNA2, and RASEF) are activated. Third, HCC patients with higher DNMT3a and TET2 have less response to sorafenib, and survive shorter, consistent with previous investigations.^44,45 Fourth, disruption of DNMT3a-TET2 crosstalk impairs resistant growth and sensitizes sorafenib^R cells to sorafinib treatment, where TSGs are re-expressed through promoter DNA hypomethylation and oncogenes are downregulated. Finally, concurrent dysfunction of DNMT3a and TET2 leads to more robust inhibition on sorafenib^R cell growth than single gene changes. Thus, DNMT3a-TET2 crosstalk serves as a new prognostic biomarker and a novel therapeutic target in overcoming HCC sorafenib resistance.

When DNA is methylated by DNMTs, the 5mC can be the substrate of TET-mediated oxidation process forming 5hmC, leading to DNA demethylation.⁸ Thus, aberrant TET activities by gene dysregulation or mutations significantly regulates cancer pathogenesis and drug resistance. Of these three TETs, TET2 frequently obtain loss-of-function mutations, which greatly influence leukemic disease,⁴⁶ but with limited impacts on solid tumors, including HCC.¹³ Although tumor suppressor role of TETs followed by increased 5hmC has been appreciated, our findings revealed that TETs are upregulated or have a trend toward upregulation in HCC compared to normal tissues. HCC patients with higher TETs live shorter than those with lower TETs. These findings are consistent with recent studies that TET2 is highly expressed in HCC tissues, and TET2 overexpression is positively correlated with a shorter survival time, although TET1 upregulation plays an oncogenic role in other cancers, like leukemia¹⁵ and breast cancer.¹⁶ Regardless, one uniqueness of our studies is the demonstration that TET2 is an important contributor to acquired sorafenib resistance, because TET2 is significantly upregulated in sorafenib^R HCC cells, and a poorer survival is noticed in sorafenib-treated HCC patients overexpressing TET2. TET2 knockdown or treatment with bobcat339 impairs sorafenib^R cell growth, and partially restores sorafenib sensitivity. Given the upregulation of TET1 and TET3, the studies whether TET1 and TET3 are essential for sorafenib resistance and how TETs are upregulated in sorafenib^R cells are warranted.

It is well known that TET2 converts 5mC into 5hmC, but few studies have revealed a negative correlation between 5mC and 5hmC abundance in cancer cells. In present study, we showed that both TET and DNMTs are upregulated, and concomitantly high level of global 5mC and 5hmC co-exist in sorafenib^R cells compared with parental counterparts. But it remains unclear whether the net outcomes of DNA methylation are more 5mC or 5hmC in sorafenib^R cells. While our promoter methylation assays revealed that certain TSGs have DNA hypermethylation in their promoter, further genome-wide DNA methylation analysis is required to define the re-distribution of 5mC or 5hmC and which genes or regions have a net outcome of 5mC or 5hmC enrichment. These thorough investigations will identify new epigenetic biomarkers for diagnosis and therapeutics in overcoming sorafenib resistance.

Finally, both DNMT3a and TET2 have DNA methylation-independent functions, serving as a scaffold protein that recruits other transcription factors to silencing target gene expression. For example, DNMT3a interacts with AML1-ETO to regulate expression of miR-193,⁴⁷ and binds HDAC to silence gene expression.⁴⁸ Further, TET2 form a complex with DNMT1 or p300 to modulate DNA methylation in certain gene promoters.³⁵ However, such methylation-independent functions of DNMT3a and TET2 in sorafenib resistance are not defined. We demonstrate that both DNMT3a and TET2 are upregulated, but they do not form a regulatory loop, because no positive correlation of their expression is observed. Knockdown of TET2 did not change the DNMT3a expression or vice versa. Actually TET2 forms a complex with DNMT3a and HDAC2 in sorafenib^R cells, and this complex binds TSG promoters to further silence TSGs in a DNA methylation-dependent manner (Fig. S7). In contrast, DNA methylation is not increased in the promoters of oncogenes CDK1, CCNA2 and RASEF, although both DNMT3a and TET2 bind these genes’ promoters. These findings are in line with these concepts that upregulation of the aforementioned oncogenes is attributed to the DNMT3a-TET2-HDAC2 complex in a DNA methylation-independent manner in resistant cells (Fig. S7). Not only do these findings identify a novel complex, DNMT3a/TET2/HDAC2, in sustaining sorafenib^R cell growth, but also these results provide a mechanistic explanation for a new functional cooperation of DNMT3a and TET2 in developing sorafenib resistance.

Our study uncovers a coordinated DNMT3a-TET2 in response to sorafenib as a hitherto unknown molecular base to initiate and sustain HCC resistance. Our data demonstrates a new mechanistic rule of aberrant epigenetic regulators in determining HCC cell fate under sorafenib-imposed selective pressure, in which the DNMT3a-TET2-HDAC2 complex modulates TSGs and cell proliferation genes through DNA methylation-dependent and -independent manners. Our results suggest an attractive prognostic biomarker and noves therapeutic target (DNMT3a-TET2-HDAC2 complex) for refractory HCC patients. Thus our findings warrant further exploration of targeting the DNMT3a-TET2 coordination as alternative approaches to counteract sorafenib resistance. Given that the efficacy of available inhibitors for epigenetic abnormality is limited in HCC therapy, and as DNMT3a-TET2 coordination exists in CSCs, disruption of DNMT3a-TET2 interplay could be used in a neo-adjuvant manner to prevent emergence of resistant oncospheres, or for eradication of sorafenib resistant cells.

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Availability of data and materials

The analyzed datasets in present study are available from the corresponding author on reasonable request.

Competing interests

The authors state no conflict of interest.

Funding

This work was supported in part by The Hormel Foundation (S.L.), National Cancer Institute (Bethesda, MD) grants R01CA248019 (S.L.), Eagle's Telethon Post Doctoral Fellowship and Paint the Town Pink fund through the Hormel Institute University of Minnesota.

Author contributions

S.L. conceived the project, designed the research and supervised the experiments conducted in the laboratories; T.C., C.Z., J.P performed the experiments and/or data analyses; T.C. analyzed sequence data and perform statistical analysis; N.T., W.T., Y.L. and S.L wrote and correct the paper. All authors discussed the results and commented on the manuscript.

Acknowledgements

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Authors' information

Shujun Liu and Yahui Liu jointly supervised this work.

Affiliations

The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA.

Tao Cheng, Changli Zhou, Jiuxia Pang

Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 2231 Sixth Street SE, Minneapolis, MN 55455.

Natalia Tretyakova

Department of Hematology & Oncology, MetroHealth Cancer Center, Case Western Reserve University School of Medicine, 2500 MetroHealth Drive, Cleveland, OH 44109, USA.

William W. Tse

Department of Hepatopancreatobiliary Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China.

Yahui Liu

Corresponding authors

Shujun Liu, Yahui Liu

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Coordinated activation of DNMT3a and TET2 in cancer stem cell-like cells initiates and sustains drug resistance in hepatocellular carcinoma

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