TET3 governs malignant behaviors and unfavorable prognosis of esophageal squamous cell carcinoma by activating the PI3K/AKT/GSK3β/β-catenin pathway

Abstract Ten–eleven translocation 3 (TET3) participates in tumorigenesis and malignant transformation by mediating DNA demethylation and specific gene activation in malignances. This study aims to elucidate its molecular function and regulatory mechanism in esophageal squamous cell carcinoma (ESCC). Stable ESCC cells that infected with TET3 overexpression (OE) and knockdown lentiviral vector had been established. The biological behaviors and molecular mechanism of TET3 were demonstrated by cell biology experiments in vitro and in vivo. Tissues from patients with ESCC were used to demonstrate the clinical value of TET3. Our findings revealed that TET3 is highly expressed in ESCC tissues and related to poor prognosis of patients with ESCC. OE of TET3 presented a significant effect on proliferation, metastatic potential, and spheroid formation of ESCC cells by activating the PI3K/AKT/GSK3β/β-catenin axis. Knockdown of TET3 could remarkably reverse these malignant phenotypes. Patients with ESCC with high TET3 expression resulted in a shorter overall survival (OS) and disease-free survival. Based on the multivariate analysis, TET3 could be an independent favorable factor for predicting OS and recurrence. The high expression of TET3 not only aggravates malignant behaviors in vitro and in vivo but also becomes a novel biomarker for clinical monitoring and individualized precision treatment for patients with ESCC.


Introduction
Esophageal cancer (EC) is a malignant tumor derived from epithelial cells, in which incidence and mortality are seventh and sixth, respectively, in global human cancers [1]. Due to the insidious onset of EC, a considerable number of patients with EC have developed to advanced stages at their first diagnosis. Postoperative metastasis and recurrence are the key factors seriously restricting the prognosis and long-term survival of patients with EC [2][3][4][5]. Histologically, EC can be divided into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma, among which ESCC is currently the main histological type in China and western countries [6]. Up to now, surgery is still the first choice for clinical radical treatment of ESCC in combination with adjuvant therapy [7,8]. Despite the continuous improvement in the clinical early diagnosis and combined treatment of EC, the prognosis is still far from satisfactorily [9]. Elucidating the underlying mechanism will undoubtedly benefits the clinical prevention and targeted therapies of ESCC.
The formation, evolution, Infiltration and metastasis of ESCC are complex biological processes involving multimolecular participation and mutual regulation, among which epigenetic variation and modification might play a crucial role [10]. Ten-eleven translocation (TET) family members are newly identified DNA demethylases including TET1, TET2, and TET3. The main biological function of TETs is converting 5-methylcytosine to generate 5-hydroxymethylcytosine, thereby mediating DNA demethylation and specific gene activation [11]. Dysregulation of TETs in several malignant tumors has been reported, including breast cancer [12], hepatocellular carcinoma [13], melanoma [14], glioma [15], renal cell carcinoma [16], and colorectal cancer [17]. Although accumulating evidence has shown that TETs are associated with tumorigenesis and progression, the specific mechanism remains highly mysterious.
In this present study, we confirmed that TET3 was overexpressed in ESCC. Further studies identified that PI3K/AKT/GSK3β/β-catenin pathway drives ESCC through TET3 regulation for stem cell-like maintenance and survival. TET3 has the potential to become a new biomarker or a therapeutic target for ESCC.

Patients and collection of tissue samples
ESCC tissue samples and paired para-cancerous tissue samples (n = 62) were enrolled from patients, which were pathologically diagnosed as primary ESCC and received surgical resection from March 2013 to January 2014 at Changhai Hospital, Navy Military Medical University (Shanghai, China). All of the patients did not receive preoperative adjuvant therapy including radiotherapy, chemotherapy, or biotherapy. The extraction of total RNA from ESCC tissues was carried out by conventional steps and detected by using the quantitative real-time PCR (RT-qPCR) assay. The extraction of total protein from 7 pairs of ESCC tissues was conducted and detected by using the western blot assay. All clinical samples were pathologically confirmed by two independent pathologists and was followed up until January 2019.

Cell culture
Human normal esophageal epithelial cell line (HEEC) was used as normal cell. ESCC cell lines Eca109 and TE1 were obtained from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China), which were conventionally cultured in RPMI 1640 (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GIBCO, USA) at 37°C in 5% CO 2 .

Spheroid formation assay
For spheroid formation, ESCC cells with different treatment groups were digested into suspensions and then seeded in ultra-low adhesion cell plates (Corning, USA) with complete medium (1 × 10 3 cells/well). The number of spheroid clusters was counted every other day until day 7.

Colony formation assay
ESCC cells (1 × 10 3 cells/well) were seeded into six-well culture plates with complete medium. Cell colonies were fixed with 4% formaldehyde and stained with crystal violet staining solution (Beyotime Biotech) after culturing for 2 weeks. The number of clones was counted by using light microscope. Three independent experiments in each group were carried out.

Cell migration and invasion assay
The experimental procedure was conducted conventionally. In wound-healing assays, ImageJ software (V1.8.0, NIH, USA) was adopted to measure the injury aera. In Transwell assay, cells that passed through upper chambers were fixed using methyl alcohol and stained with crystal violet staining solution (Beyotime Biotech). Three independent experiments in each group were carried out.

Animal experiment
Animal experiments have been complied with the permission approved by the Committee on Ethics of Medicine, Navy Military Medical University. All animals were raised under SPF condition, all experimental procedures were in accordance with the criteria. Twenty-six-week-old BALB/c male nude mice were randomly divided into four groups (five mice/each group): control group (EGFP), TET3-OE group (OE), control group (pLKO), and TET3-knockdown group (KD1). In subcutaneous model, ESCC cells (1 × 10 7 cells/mouse) were subcutaneously injected into each mouse and raised for 42 days. After that, mice were sacrificed painlessly and tumor volume and weight were calculated. In the lung metastasis model, ESCC cells (5 × 10 5 cells/mouse) were injected into the tail vein and raised for 30 days. Mice were sacrificed and dissected to assess the number of metastatic nodules in lungs.

Statistical analysis
GraphPad Prism 5.0 software and SPSS 22.0 software were used for statistical analysis. Data were presented as mean ± standard deviation. Statistical differences between groups were tested by using Student's t-test or one-way analysis of variance test. Kaplan-Meier survival method was used to analyze survival condition. P < 0.05 indicated that the difference was statistically significant. 3 Results

TET3 is overexpressed in ESCC
Based on the data from UALCAN (Data from TCGA database) (http://ualcan.path.uab.edu), we compared the mRNA expression of TET3 between ESCA tissues (n = 184) and normal tissues (n = 11). The results revealed that the expression of TET3 was elevated in tumors than that in normal tissues (P = 0.00252) ( Figure A1(a)). We further focus on the correlation between the mRNA levels of TET3 and the survival time of patients with ESCA by using publicly available datasets (UALCAN).
The results indicated that patients with ESCA with the high expression of TET3 mRNA were correlated with shorter survival time (P = 0.048) ( Figure A1(b)). To confirm the above conclusion, we detected the protein expression of TET3 in 62 ESCC samples and para-cancerous tissues by using IHC. The results suggested that TET3 was located in cytoplasm and nuclei, which were elevated in ESCC tissues compared with paired para-cancerous tissues (Figure 1a and b). We collected seven pairs of freshly surgically resected ESCC tissues and extracted total RNA and protein using the RT-qPCR assay and the western blot analysis, respectively. Our results revealed that the protein and mRNA levels of TET3 were dramatically up-regulated in ESCC tissues (Figure 1c-e). Meanwhile, the protein and mRNA expression of TET3 in ESCC cell lines (TE1 and Eca109) were upregulated compared with normal HEEC (Figure 1f-h). According to these results, we confirmed that TET3 was abnormally up-regulated in ESCC.

TET3 promotes the proliferation and metastasis of ESCC cell lines in vitro and in vivo
To explore the biological function of TET3, endogenous OE and knockdown cell sublines were established in TE1 and Eca109. The exogenous TET3-EGFP was stably expressed in TE1 (OE) and Eca109 (OE). The transcription level of TET3 was stably down-regulated by two different shRNA vectors in TE1 (KD) and Eca109 (KD). Fluorescence microscopy showed that TET3 was located in cytoplasm and nuclei ( Figure A2(a)). The OE and knockdown level of TET3 in ESCC cells were confirmed by the western blot analysis ( Figure A2(b)). Colony formation, Transwell, and wound-healing assays were employed to evaluate the proliferation and motility of TET3-OE/KD Eca109 and TE1 cells. Our results found that the OE of TET3 significantly promoted the ability of proliferation and the motility of ESCC cells, while knockdown of TET3 exhibited the opposite outcomes (Figure 2a-c).
Animal models by subcutaneous and tail vein injection in nude mice were established to evaluate the function of TET3 on tumorigenicity and metastatic potential of ESCC cells in vivo. The results revealed that the tumor volume and weight in TET3-OE group were significantly larger than those in the control group (Figure 3a-c). By contrary, the tumor volume and weight in TET3-KD group were significantly decreased compared with those in the control group (Figure 3d-f). HE staining showed that the number of metastatic nodules in lung tissues of TET3-OE group was statistically more than that in the control group, and TET3-KD group had the least number of lung metastatic nodules (Figure 3g-j). Our present results suggested that the high expression of TET3 could promote the proliferation and metastasis potential of ESCC cells in vitro and in vivo.

TET3 promotes spheroid formation of ESCC cells in vitro
To further explore the effect of TET3 on the maintenance of stem cell-like characteristics in ESCC cells, we examined the expression of candidate cancer stem cell (CSC) markers. Three-dimensional spheroid formation assay was used to evaluate the spheroidizing potential of ESCC cells. In the TET3-OE group, the ability of tumor cells to form spheroids was increased. Down-regulation of TET3 inhibited the formation of spheroids (Figure 4a). The stem cell markers in ESCC cell spheroids were measured by immunofluorescence staining, and we found that the stem cell markers, including EpCAM, βcatenin, and CD133 were positive (Figure 4b). While knockdown of TET3 down-regulated the protein level of β-catenin and CD133 in spheroids. These results demonstrated that the spheroids have the characteristics of stem cells (Figure 4b). Down-regulation of TET3 could inhibit the formation of ESCC cell spheroids and decreased the expression level of CSC markers. In summary, high expression of TET3 could promote the formation of spheroids and the maintenance of stem celllike characteristics in ESCC cells.

TET3 enhances the malignant transformation of ESCC cells by activating the PI3K/AKT/GSK3β/β-catenin axis
It has been widely recognized that PI3K/AKT signaling pathway could participate in the malignant progression of a variety of human tumors by regulating the growth of tumor cells, invasion, metastasis, and angiogenesis and maintaining the characteristics of tumor stem cells [18,19].
GSK3β is a crucial substrate in the PI3K/AKT signaling pathway and regulates several physiological processes, such as the activity of mitochondria [20]. Based on IHC results of serial tissue sections of ESCC, the protein level of p-AKT (T308), p-AKT (S473), EpCAM, and β-catenin was up-regulated; meanwhile, p-GSK3β (Tyr216 + Tyr279) and p-GSK3β (Ser9) are relatively low (Figure 5a). In addition, the up-regulation of TET3 led to a significant increase in p-AKT (T308), p-AKT (S473), and β-catenin while inhibited the expression of p-GSK3β (Tyr216 + Tyr279) and p-GSK3β (Ser9) in ESCC cells. Knockdown of TET3 expression led to the activation of the expression of p-GSK3β (Ser9); meanwhile, it down-regulated p-AKT (T308), p-AKT (S473), and β-catenin, but the expression changes in p-GSK3β (Tyr216 + Tyr279) were not obvious (Figure 5b). According to these results, we could infer that TET3 inhibits the phosphorylation level of GSK3β (especially on Ser9 site) through the activated PI3K/AKT signaling pathway, enhancing the stability of β-catenin in cancer cells and subsequently participating in downstream gene transcription related to cell proliferation and metastasis.

High expression of TET3 leads to poor prognosis for patients with ESCC
The expression pattern of TET3 and clinicopathological indicators of patients with ESCC was analyzed. These results indicated that the gender, age, degree of differentiation, and TMN staging were not correlated with the OE of TET3 of patients with ESCC ( Table 1). The elevated expression of TET3 was significantly correlated with lymph node metastasis (P = 0.047). According to the staining scores of TET3, we divided 62 patients with ESCC into the high expression group (n = 41) and the low expression group (n = 21). The patients were followed up from 6 months to 60 months, and the median time was 42.2 ± 18.1 months. Kaplan-Meier survival analysis was carried out to analyze the expression of TET3 on the clinical prognosis of patients with ESCC. As presented in Figure 5c, high expression of TET3 was related to lower overall survival (OS) rate (P = 0.048) and disease-free survival (DFS) rate (P = 0.023) compared with the TET3-low group. Obviously, OE of TET3 was significantly correlated to the poor prognosis and survival of patients with ESCC. Based   I-II  25  13  12  III-IV  37  28  9 TET3: ten-eleven translocation 3; ESCC: esophageal squamous cell carcinoma; TNM: tumor, lymph node, and metastasis.
on the univariate analysis, we selected lymph node metastasis (P = 0.001), TMN staging (P = 0.003), and TET3 expression (P = 0.001) for multivariate analysis. The results revealed that TET3 has the potential to be an independent favorable factor for OS and recurrence (P = 0.001, Table 2). Collectively, clinical analysis suggested that elevated expression of TET3 may promote the malignant transformation of ESCC and is closely related to poor prognosis.

Discussion
In our present study, we found that TET3 was abnormally overexpressed in ESCC cells and tissues, which could be acted as an oncogene by facilitating malignant progression and predicting poor prognosis. Tumor recurrence and metastasis are multi-step processes, which usually accompanied by specific genomic, proteomic, metabolic, and epigenetic changes [21][22][23]. In recent decades, accumulating novel biomarkers could be used for early diagnosis and clinical therapy, accompanied with studies focus on the mechanism of tumorigenesis, and progression has made remarkable achievements. However, it is regrettable that some clinical challenges, such as the precise molecular mechanism of tumorigenesis, proliferation, and metastasis, maintenance of tumor stem-like characteristics, acquired drug resistance including chemotherapeutic drugs, and radiotherapy, still have not been well solved.
ESCC is one of the death-related tumors in China [24]. Postoperative recurrence and metastasis of ESCC are the crucial factors restricting the long-term survival of patients [25]. Based on the sequencing data by our group, screening and analyzing the public databases (http://ualcan.path.uab.edu), we locked TET3 as the target molecular. We investigated the function and possible mechanism of TET3 in ESCC, for the purpose of elucidating the molecular mechanism, regulatory function, and the clinical value in ESCC.
As we know, TET family members were dysregulated in various tumors, which play the function of oncogene or tumor suppressor gene. TET3 was decreased in ovarian cancer and silencing of which could inhibit malignant transformation of cancer cells [26]. But in acute myeloid leukemia, TET3 expression was significantly increased and could be used as an independent prognostic factor [27]. Therefore, the biological function of TET3 may be tissue specific. Combined with the information of the public database and our present results, we validated that TET3 was dramatically overexpressed in ESCC, which was consistent with Murata's experimental results [28]. That is to say, TET3 could serve as an oncogene in the initiation and progression of ESCC. Furthermore, molecular mechanism study found that up-regulation of TET3 could elevate the proliferation and metastasis ability of ESCC cells. This phenomenon has also been verified in nude mice. Deprivation of TET3 expression remarkably inhibited the proliferation and metastatic ability of ESCC cells in vitro and in vivo. Meanwhile, based on the clinical data, we got a consistent result that elevated level of TET3 was significantly correlated with poor prognosis of patients with ESCC.
Studies have shown that OE of TET3 contributes to inducing and maintaining the CSC-like phenotype [29][30][31].
Recent findings revealed that, as a functional approach, spheroid formation is particularly useful to enrich the potential CSC subpopulations [32,33]. In this study, we found that OE of TET3 could enhance the spheroidization and the expression level of CD133 and β-catenin in ESCC cells in vitro. Down-regulation of TET3 decreased the ability of spheroid formation, meanwhile the expression of above stem cell-related markers was also significantly inhibited. These CSC-like cells may have significant growth advantages and the ability to resist survival pressure, even led to tumor proliferation and metastasis under specific physiological condition.
It has been widely recognized that PI3K/AKT is a classic signaling pathway, which participates in malignant progression of a variety of human malignances by regulating proliferation of tumor cells, invasion and metastatic potential, angiogenesis and maintaining the characteristics of tumor stem cells [18,19]. GSK3β is a crucial substrate in the PI3K/AKT signaling pathway and regulates the activity of mitochondria [20]. Abnormal phosphorylation level of GSK3β could affect Wnt/β-catenin/T-cytokine (TCF) signals and mediate epithelial mesenchymal transformation (EMT) process by participating in the transcriptional regulation of snail (EMT-related transcription factor) expression [34,35]. However, activation of PI3K/AKT signaling pathway could suppress GSK3β phosphorylation at the Ser9 site, which enhances the stability of β-catenin in cells, promotes its nuclear translocation, and eventually participates in the transcriptional regulation of downstream cell proliferation and metastasis-related genes [20,36]. Our present study revealed that the expression of TET3 was significantly positively correlated with βcatenin and the phosphorylation level of AKT (T308 and S473) was negatively correlated with the phosphorylation level of GSK3β (Ser9). These results confirmed that TET3 could inhibit the phosphorylation level of GSK3β (mainly through Ser9 site) by activating the AKT signaling pathway, eventually to stabilize and promote the β-catenin expression in ESCC cells. Taken together, our data suggested that TET3 may act as an oncogene in ESCC. Elevated expression of TET3 could significantly facilitate the malignant progression, including proliferation, metastasis, and maintenance of stem cell characteristics in ESCC cells via PI3K/AKT/ GSK3β/β-catenin pathway. The importance of TET3 in prognostic survival has also been validated in patients with ESCC. TET3 is expected to become a novel molecular marker for clinical diagnosis, treatment, and monitoring prognosis in the future.