JMJD3 suppresses tumor progression in oral tongue squamous cell carcinoma patients receiving surgical resection

Patient selection

Between January 2007 and December 2016, a total of 1,143 OTSCC patients treated at Kaohsiung Chang Gung Memorial Hospital were retrospectively reviewed. First, we excluded patients who had a history of any second primary malignancy or distant metastasis. Second, we also excluded those who received neoadjuvant chemotherapy, radiotherapy, or chemoradiotherapy. Third, only patients who underwent curative surgical resection were enrolled. Fourth, only patients with complete medical records were included. Finally, a total of 156 OTSCC patients were identified.

Tumor grade were classified as grade 1 (well differentiation), grade 2 (moderate differentiation) and grade 3 (poorly differentiation) by the pathologists based on how abnormal the tumor cells and the tumor tissue look under a microscope. The definition of smokers included “current smokers” who have smoked more than 100 cigarettes in their lifetime and has smoked in the last 28 days, and “ex-smokers” that means they have smoked more than 100 cigarettes in their lifetime but have not smoked in the last 28 days.

Immunohistochemistry

The formalin fixed paraffin wax embedded OTSCC tissue was sectioned to be 4 µm in thickness for each patient. First, deparaffinization by incubation in a dry oven at 60 °C for 1 h, antigen retrieval in 10 mM citrate buffer (pH 6.0) in a hot water bath (95 °C) for 20 min, and peroxidase blocking with 0.3% hydrogen peroxide for 5 min. After that, the specimen was reacted with primary antibody against JMJD3 (Abcam, ab38113, 1:100, Cambridge, UK), and then with a ready-to-use visualization reagent consisting of goat secondary antibody. Then, tissue was incubated with polymer for 8 min and followed by 3–3′-diaminobenzidine for 10 min and hematoxylin for counterstaining. The slide of normal colon mucosa was used as a positive control; for the negative control, primary antibodies were omitted. The scores of these slides were investigated by 2 pathologists (WT Huang and SL Wang) blinded to clinicopathologic features or prognosis. The method to score the expression of JMJD3 was determined according to previous published study (Shen et al., 2012).

The proportions of JMJD3-expressing tumor cells were scored by immunoreactive score (IRS) system which is calculated by the product of the multiplying the staining intensity (no staining = 0, weak staining = 1, moderate staining = 2, strong staining = 3) and percentage of positive stained cells (0% = 0, 1–10% = 1, 11–50% = 2, 51–80% = 3, 81–100% = 4), resulting in IRS scores between 0 (no staining) and 12 (maximum staining) (Remmele & Stegner, 1987). A specimen with a sum score > 6 was regarded as high expression.

Cell lines and culture

SAS and Cal 27, two cell lines of OTSCC, were purchased from American Type Culture Collection, and cultured in the Dulbecco’s modified Eagle’s medium, containing 10% of fetal bovine serum. Cells were cultured at 37 °C. We had performed the short tandem repeat profiling of these cell lines and the last date of authentication was 02 July 2020.

Western blot analysis

GSK-J4, a UTX-specific pharmacological inhibitor (SML0701, Sigma-Aldrich, St. Louis, Missouri, USA), reconstituted in DMSO, with different concentrations (0, 2, 4, 8 µM) were given to these cells for 48 h. Whole-cell lysates of GSK-J4 treated cells were extracted using the fixture of RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) and subjected to western blot analyses. The membranes were incubated with primary antibodies against JMJD3 (ab169197, 1:2000, Abcam, Cambridge, MA, USA), Rb (#9309, 1:1000, cell signaling, Danvers, Massachusetts, USA), phosphorylated Rb (pRb) (ab173289, 1:1000, Abcam, Cambridge, UK), p21 (#2947, 1:1000, Cell Signaling, Danvers, Massachusetts, USA) and β-actin (A5441, 1:10000, Sigma-Aldrich, St. Louis, Missouri, USA) at 4 °C overnight. After that, the membranes were incubated with secondary antibodies for one hour at room temperature to reveal the immune detection. The protein signals were detected with Western Lightning Chemiluminescence Reagent Plus (Amersham Biosciences). All the experiments were repeated at least three times with similar results.

Statistical analysis

Three independent experiments were performed and similar results were documented. The statistical analysis was performed using SPSS software (International Business Machines Corp, New York, USA). The Chi-square test was used to examine the difference of categorical variables. Disease-free survival (DFS) was calculated from the time from surgery to recurrence of tumor or death from any cause. Overall survival (OS) was defined as the duration between the time of diagnosis of OTSCC to death or last living contact. The Kaplan–Meier method was used for univariate analysis, and the log-rank test was performed to assess the difference. Multivariate analysis was examined using Cox proportional hazards model to identify the independent prognostic factors. A two-tailed p value of < 0.05 was considered to statistically significant.

Ethics statement

The ethic approval for this study was obtained from the Chang Gung Medical Foundation Institutional Review Board (201901388B0). All procedures performed in studies were in accordance with the ethical standards of the institutional research committee and the World Medical Association Declaration of Helsinki. Written informed consent was waived by the Chang Gung Medical Foundation Institutional Review Board.

Patient characteristics

We identified 156 OTSCC patients who underwent surgical resection at Kaohsiung Chang Gung Memorial Hospital between January 2007 and December 2016. All of the 156 OTSCC patients had an Eastern Cooperative Oncology Group performance status ≤1. In our study, 143 patients were male and 13 patients were female, and the median age was 53 years (range: 26–86 years). The study included 127 patients (81%) who had a history of cigarette smoking, 124 patients (80%) who had a history of alcohol consumption, and 116 patients (74%) who had a history of betel-nut chewing. Forty-five patients (29%) were diagnosed to have T1 status, 49 patients (32%) with T2 status, 13 patients (8%) with T3 status, and 49 patients (31%) with T4 status. Regarding the tumor N status, 86 patients (55%) had N0, 24 patients (15%) had N1, 43 patients (28%) had N2, and three patients (2%) had N3. There were 34 patients (22%) in stage I, 30 patients (19%) in stage II, 24 patients (15%) in stage III, 61 patients (39%) in stage IVA, and seven patients (5%) in stage IVB. Tumor grade analysis revealed that 91 patients (58%) were in grade 1, 59 patients (38%) were in grade 2, and six patients (4%) were in grade 3. Adjuvant radiotherapy or chemoradiotherapy were performed based on adverse risk factors, such as extracapsular extension, positive surgical margin, pathological T3 or T4 status, presentation of multiple lymph node involvement, lymphovascular or perineural invasion.

In the current study, the median follow-up period was 87.9 months for the 71 living survivors and 59.2 months (range: 1.0–112.1 months) for all 156 patients. The five-year DFS and OS rates were 46.2% and 50.0%, respectively. Detailed information is shown in Table 1.

JMJD3 and clinical outcome

Results of the immunohistochemical staining of JMJD3 are shown in Fig. 1. Among the 156 patients, 73 patients (47%) had high expression of JMJD3 and 83 patients (53%) had low expression of JMJD3. The two groups were compared for baseline characteristics, and no significant difference was observed for age, sex, histologic grade, vascular invasion, surgical margin, alcohol consumption, cigarette smoking and betel-nut chewing. Patients with low expression of JMJD3 had a higher percentage of pathological T state, pathological N status, tumor stage, perineural invasion, and extracapsular extension than those with high expression of JMJD3. The comparison results are shown in Table 2.

With respect to DFS, age, sex, histologic grade, surgical margin, alcohol consumption, and betel-nut chewing did not meet statistical significance in the univariate analysis. The 62 patients with pathological T1-2 were found to have a better five-year DFS rate compared with the 94 patients with pathological T3-4 (53% versus 36%, P = 0.007); meanwhile, the 86 patients without nodal metastasis had a superior five-year DFS rate than the rest 70 patients with nodal metastasis (57% versus 33%, P < 0.001). A significant five-year DFS rate was mentioned in the 64 patients who had pathological stage I–II than in the 92 patients who had pathological stage III–IV (59% versus 37%, P = 0.001). One hundred thirty-one patients without vascular invasion were found to have a superior five-year DFS rate compared to 25 patients with vascular invasion (50% versus 28%, P = 0.028), and a better five-year DFS rate was observed in 87 patients without perineural invasion than in the other 69 patients with perineural invasion (55% versus 35%, P = 0.003). A significantly improved five-year DFS rate was found in 119 patients without extracapsular extension compared to that in 37 patients with extracapsular extension (52% versus 27%, P < 0.001); in addition, 29 patients without cigarettes smoking had a better five-year DFS rate in comparison with 127 smokers (66% versus 42%, P = 0.028). Seventy-three patients who had high expression of JMJD3 had a better five-year DFS rate than the rest 83 patients with a low expression of JMJD3 (59% versus 35%, P = 0.001, Fig. 2A). Multivariate analysis showed that pathological T1-2 status (P = 0.038, hazard ratio (HR): 0.63, 95% confidence interval (CI) [0.40–0.97]), grade 1 (P = 0.012, HR: 0.58, 95% CI [0.38–0.88]), no extracapsular extension (P = 0.002, HR: 0.48, 95% CI [0.30–0.76]), and high expression of JMJD3 (P = 0.011, HR: 0.57, 95% CI [0.37–0.88]) were the independent prognostic factors of a superior five-year DFS rate.

In the analysis of OS, the univariate analysis revealed that there were no significant differences in age, sex, histologic grade, alcohol consumption, and betel nut chewing. The 62 patients who had pathological T1-2 were found to have a better five-year OS rate compared with the 94 patients who had pathological T3-4 (59% versus 37%, P = 0.002); meanwhile, the 86 patients without nodal metastasis had a superior five-year OS rate than the rest 70 patients with nodal metastasis (61% versus 37%, P < 0.001). The 64 patients who had pathological stage I–II were found to have a significantly better five-year OS rate than the 92 patients who had pathological stage III–IV (63% versus 41%, P = 0.001). The 131 patients without vascular invasion were mentioned to have a better five-year OS rate than the 25 patients with vascular invasion (54% versus 28%, P = 0.028), and a better five-year OS rate was observed in the 87 patients without perineural invasion than in the other 69 patients with perineural invasion (58% versus 41%, P = 0.012). The 119 patients without extracapsular extension had a significantly superior five-year OS rate compared to the 37 patients with extracapsular extension (56% versus 30%, P < 0.001); meanwhile, a superior five-year OS rate was observed for the 145 patients without surgical margin compared with the 11 patients with a surgical margin (52% versus 27%, P = 0.016). The 29 patients without cigarettes smoking had a superior five-year OS rate compared to the 127 smokers (72% versus 45%, P = 0.016). The 73 patients who had high expression of JMJD3 had a better five-year OS rate than the other 83 patients with low expression of JMJD3 (63% versus 39%, P = 0.001, Fig. 2B). In the multivariate analysis, pathological T1-2 status (P = 0.030, HR: 0.60, 95% CI [0.37–0.95]), grade 1 (P = 0.007, HR: 0.54, 95% CI [0.34–0.85]), vascular invasion (P = 0.034, HR: 0.55, 95% CI [0.32–0.95]), no extracapsular extension (P = 0.003, HR: 0.48, 95% CI [0.30–0.78]), and high expression of JMJD3 (P = 0.013, HR: 0.57, 95% CI [0.36–0.88]) were the independent prognostic factors of a better five-year OS rate. Survival outcome results of the univariate and multivariable analyses are shown in Tables 3 and 4, respectively.

Inhibition of JMJD3 by GSK-J4

In this in vitro study, western blot analyses were done to determine the expression of JMJD3, Rb, and p21 in the OTSCC cell lines which were treated with different concentrations of GSK-J4. Rb is a well-known tumor suppressor gene and p21 is a potent cyclin-dependent kinase inhibitor (Kent & Leone, 2019; Sherr & McCormick, 2002). Our data demonstrated that the expression of JMJD3 was decreased in GSK-J4-treated cell lines compared to that in the control cells; in addition, the expression of Rb, pRb and p21 were inhibited. The results are shown in Fig. 3.