Epigenetic identification of mitogen-activated protein kinase 10 as a functional tumor suppressor and clinical significance for hepatocellular carcinoma

Tumor cell lines

A series of HCC cell lines (Hep3B, HepG2, huH1, huH4, huH6, huH7, PLC/PRF/5, SNU387, SNU398, SNU423, SNU449, and SNU475) were maintained in Cancer Epigenetics laboratory, Department of Clinical Oncology, PWH, The Chinese University of Hong Kong, Shatin, Hong Kong and cultured in RPMI or DMEM (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Gaithersburg, MD, USA) and incubated at 37 °C in a humidified chamber containing 5% CO₂.

Tissues of HCC

A total of 18 fresh primary HCC tissues were collected from patients who underwent hepatectomy for HCC in 2009 at the First Affiliated Hospital of Chongqing Medical University (Chongqing, China). Among them,10 has the matched non-tumor liver samples, obtained from at least 5 cm distant from the tumor. All of the fresh tissues were processed routinely for histopathological assessment and immediately immersed in liquid nitrogen after surgical resection.

Along with the paraffin-embedded tissues, a retrospective cohort of 59 patients with HCC was enrolled in the current study. These patients underwent hepatectomy at the First affiliated hospital of Chongqing Medical University (Chongqing, China) between June 2005 and December 2009. All patients were diagnosed with primary HCC by two pathological doctors, and there was no previous radio- or chemotherapy before surgery. The study was carried out in accordance with the Declaration of Helsinki and the protocol was approved by Ethics Committee of Chongqing Medical University (Chongqing, China) and written informed consent was obtained from each patient involved in the study.

Immunohistochemistry

IHC staining was carried out by using Ultrasensitive TMS-P (MXB, Fuzhou, China). The primary antibody against MAPK10 was obtained from Abcam (Abcam, Cambridge, MA, USA) and incubated the sections at a dilution 1:100. The staining intensity was scored as 0 (absent expression), 1 (weak expression), 2 (moderate expression), and 3 (strong expression) and the percentage of positive staining as 0 (0–9%), 1 (10–25%), 2 (26–50%) or 3 (51–100%) (Jiang et al., 2015). The final score was obtained by multiplying as the following: the staining intensity score × the percentage of positive staining and ranked from 0 to 9. So, the Mapk10 expression of HCC was defined as “−”, “+”, “++” and “+++” corresponding to score 0, 1–3, 4–6 and 7–9 respectively. For further clinical analysis, we divided the HCC into two subgroups: the “Mapk10-negative” subgroup (“−”) or “Mapk10-positive” subgroup "+−+++". The IHC staining score was performed in a double-blind manner by two pathologists to minimize the observational bias.

Semiquantitative RT-PCR

RT-PCR was conducted to detect the expression of Mapk10 in HCC cell lines. RT-PCR was performed for 32 cycles with hot-start Go-Taq (Promega, Madison, WI, USA) (Tao et al., 2002). The primers are listed in Table 1.

Western blot

Primary antibodies against Mapk10 (1:000, Abcam, USA) and PARP (1:000, Abcam, Cambridge, MA, USA) were used. At 48 h post-transfection, the cells were collected and lysed in RIPA lysis buffer (Beyotime, Haimen, China). Total protein was dissociated by SDS-PAGE, transferred onto PVDF membrane, and immunoblotted. The immunoreactive bands were exposed by the ECL detection system.

Methylation-specific PCR

After DNA bisulfite treatment, MSP was performed in the HCC cell lines as well as in the primary HCC tumors and adjacent non-tumor tissues as described previously (Tao et al., 2002). The MSP primers detecting the methylated or unmethylated alleles are MAPK10m3/MAPK10m5 and MAPK10u3/MAPK10u5, respectively (Table 1) (Ying et al., 2006). These primer pairs have been tested previously that they did not amplify any unbisulfited DNA. MSP was performed for 40 cycles with AmpliTaq-Gold (Applied Biosystems, Waltham, MA, USA) (Tao et al., 2002). YccB1, a cell line of breast carcinoma with hypermethylated MAPK10 which has been demonstrated in the previous literature was used as the positive control of MAPK10 methylation (Ying et al., 2006).

5-aza-2′-deoxycytidine (Aza) and Trichostatin A (TSA) treatment

A density of 1 × 10⁵ HCC cells/mL were allowed to grow overnight. Then, fresh medium containing Aza at a final concentration of 10 mM (Sigma-Aldrich, St. Louis, MO, USA) was used to replace the culture medium. The cells were treated with Aza for 72 h, followed by the histone deacetylase inhibitor TSA at 100 nM for another 24 h. Finally, the cells were collected for methylation-specific PCR (MSP) and the gray value of bands was analyzed by Image J software.

Gene cloning and plasmid construction

The Mapk10 recombinant plasmids were purchased from GeneCopoeia (Rockville, MD, USA). These were termed as OmicsLink™ expression clone (EX-A1150-M29) and pcDNA3.0(+)-Flag-MAPK with the accurately confirmed sequence and orientation.

Subcellular localization

A density of 5 × 10⁴ of Hep3B or HepG2 cells were planted on coverslips in a 6-well plate. Then, the cells were transfected with pcDNA3.0(+)-Flag-Mapk10 using Lipofectamine 2000 (Invitrogen, Carlsbad CA, USA). After 24 h, the cells were fixed with 4% paraformaldehyde phosphate buffer solution and incubated with anti-Flag M2 monoclonal antibody (Sigma, St. Louis, MO, USA), followed by FITC-conjugated rabbit anti-mouse IgG F(ab)2 antibody (Sigma, St. Louis, MO, USA). Hereafter, cell nuclei were stained with DAPI (Thermo, Waltham, MA, USA) and images were captured by Leica TCS SP2 AOBS confocal laser-scanning microscope (Mannheim, Germany).

Colony formation assay

1 × 10⁵ Hep3B and HepG2 cells/well were seeded in a 12-well plate and transfected with Mapk10 plasmid or control vector, using Lipofectamine 2000. After 48 h, the cells were seeded into a 6-well plate, and subjected to G418 (0.4 mg/mL) for 10–14 days, with the medium refreshed every 2 days. The surviving colonies were enumerated after Giemsa staining. All the experiments were conducted three times in triplicate wells.

Apoptosis assay

The cell apoptosis and viability were evaluated using the Annexin V-PE Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) by flow cytometry. Apoptosis was always graded as viable, early apoptotic, necrotic and late apoptotic, which corresponds to Annexin V-/7-AAD-, Annexin V+/ 7-AAD-, Annexin V-/7-AAD+ and Annexin V+/7-AAD+. Hep3B and HepG2 cells were transfected with Mapk10 plasmid or the control vector and harvested after 48 h. The GFP-positive cells were selected, and the apoptotic status was assessed by Annexin V-PE and 7-AAD staining. Both early and late apoptotic cells were considered for relative apoptotic alterations. Apoptosis was also depended on the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. At 48 h post-transfection, the apoptotic cells were detected using the In Situ Cell Death Detection Kit POD (Roche, Burlington, NC, USA), wherein the DNA fragmentation was determined and visualized by a fluorescence microscope. All the experiments were repeated 3 times.

XTT proliferation assay

Hepatocellular carcinoma is resistant to systemic chemotherapy and 5-FU is one of the most common drugs for HCC. Herein, we assessed whether Mapk10 could enhance the chemotherapeutic effects of 5-FU on HCC cells. XTT Cell Proliferation Assay kit (Beyotime, Haimen, China) was used to determine the chemotherapy-induced cytotoxicity on HCC cells. Mapk10 or control vector was transfected into HCC cells, respectively and treated with 5-FU at different concentrations (0, 6.25, 12.5, 25, 50, 100, and 200 µg/mL) for 72 h. Subsequently, the relative number of viable cells between Mapk10-transfected group and control vector-transfected group was compared and expressed as a percentage using the following formula: cell viability (%) = A450 of cells treated with 5-FU/A450 of cells without 5-FU treatment. Three independent experiments were performed, each in duplicate.

Statistical analysis

The clinicopathological features in MAPK10-positive patients and MAPK10-negative patients were compared using the Pearson’s chi-squared test. Kaplan–Meier plots and log-rank tests were used for the survival analysis. Student’s t-test was used to compare the differences in the effect of MAPK10 expression on colony formation, cell apoptosis, and cell proliferation. SPSS 19.0 was used for data analysis. A P-value <0.05 was deemed as statistically significant.

The protein expression of MAPK10 in clinical samples of HCC and its clinical significance in HCC patients

The cohort of 59 patients (Table 2) consisted of 77.9% males with a median age of 49.94 (range, 21–77) years. Chronic HBV carriers accounted for the majority (66.2%). The median AFP (α-fetoprotein) value was 5,181.8169 (range 2.8–96,401) ng/ml. Among all the samples, 46 pairs were eligible for the comparison of the expression of mapk10 between HCC and adjacent non-tumor tissues by IHC. All of the 59 patients with follow-up data were well qualified for the clinical correlation analysis and OS analysis, with the median follow-up for 13.0 (range 2–46) months.

As for 46 paired paraffin-embedded tissues, all of the adjacent non-tumor tissues show the positive expression of MAPK10, whereas 29 HCC tissues show the negative expression, the negative expression rate of MAPK10 between the HCC and the adjacent non-tumor tissues was significant (P < 0.0001) (Fig. 1). The clinicopathological correlation analysis showed that the negative expression of MAPK10 in HCC was significantly associated with higher serum AFP (p = 0.05), more microsatellite nodules (P = 0.025) and advanced tumor stage (P = 0.001) (Table 2).

Downregulated MAPK10 expression may indicate poor prognosis in HCC patients

Prognostic significance of MAPK10 in HCC was also studied in this cohort of 59 patients with follow-up data, which indicated that the expression of MAPK10 was extremely correlative with OS of patients (log rank = 7.123, P = 0.008): the median OS time in MAPK10-negative subgroup (n = 23) and MAPK10-positive subgroup (n = 36) were 10.86 months (95% CI [5.927–15.792]) and 24.040 months (95% CI [17.511–30.568]) respectively (Fig. 2). These data suggested that MAPK10 could be an indicator for the prognosis of HCC patients.

Expression profiling of Mapk10 in HCC cell lines

Previous studies found a deletion at 4q21.3 in two esophageal carcinoma cell lines and found that Mapk10 was located in this deleted region with a 633bp bidirectional promoter, which was a typical CpG island, as well as that Mapk10 was expressed in all normal adult tissues (Ying et al., 2006). However, semi-quantitative RT-PCR in our experiments revealed a frequently reduced or silenced expression of Mapk10 in HCC cell lines (67%, 8/12) (Fig. 3). Together, the above results showed that Mapk10 is a widely expressed gene but frequently disrupted in multiple HCC cell lines.

Frequent inactivation of Mapk10 by promoter CpG methylation

In fresh primary tumors, the Mapk10 promoter was frequently methylated in 12/18 (66%) HCC tissues, while reduced or no methylation was detected in the adjacent non-tumor tissues (Fig. 4A), suggesting that the methylation of Mapk10 is tumor-specific. Furthermore, we detected Mapk10 methylation in HCC cell lines using MSP and found that the methylation of Mapk10 is associated with the dysregulated or silenced expression of the gene shown in RT-PCR (Figs. 4B and 4C).

Pharmacological and genetic demethylation restores Mapk10 expression

Two HCC cell lines, Hep3B and HepG2, were subjected with DNA methyltransferase inhibitor Aza combined with histone deacetylase inhibitor Trichostatin A (TSA). The treatment restored the Mapk10 expression, accompanied by decreased methylated promoter alleles (HepG2) or increased unmethylated alleles (Hep3B) (Fig. 5). The above results indicated that promoter methylation directly mediated the transcriptional silencing of Mapk10.

The frequent silencing of Mapk10 by hypermethylation in HCC cell lines rendered Mapk10 as a putative tumor suppressor. Furthermore, we evaluated the growth characteristics of the cells overexpressing Mapk10 by colony formation assay. HepG2 and Hep3B cell lines with silenced Mapk10 were transfected with Mapk10 recombinant plasmids, and the number of colonies was enumerated after 10–14-day culture under G418 selection. The subcellular localization of MAPK10 was examined by indirect immunofluorescence staining, which indicated that Flag-tagged MAPK10 was localized in the nucleus after the transfection into HCC cells (Fig. 6A) and the western blot detection of MAPK10 protein expression also confirmed the success of transfection (Figs. 6B and 6C). The ectopic expression of Mapk10 markedly decreased the colony formation efficiency of Hep3B and HepG2 cells (34% and 40% respectively, **P < 0.01), indicating the growth inhibitory activity of Mapk10 (Figs. 6D and 6E).

Ectopic expression of Mapk10 induces apoptosis

To explore the mechanism of the tumor suppression by Mapk10, we performed apoptosis assay using Annexin V-PE and 7-AAD double staining. The percentage of Annexin V (+/−) and 7-AAD (+/−) cells in GFP-positive cells by flow cytometry was determined. In Hep3B cells and HepG2 cells, the ectopic Mapk10 expression resulted in a significant increase in the number of apoptotic cells as compared to the control (P < 0.05) (Fig. 7A). Furthermore, the apoptotic induction was assessed by TUNEL assay at the individual cell level. Green fluorescent signals, characteristic of cell apoptosis, were presented in Mapk10-transfected cells but less in control vector- transfected cells (Fig. 7B). Increased protein level of cleaved-PARP was also shown in HepG2 and Hep3B cells after MAPK10 transduction (Fig. 7C).

Mapk10 enhances the chemosensitivity of HCC cells to 5-FU

The efficacy of chemotherapeutic drugs is dependent on their ability to trigger apoptosis (Jiang et al., 2015). Since our studies showed that Mapk10 induces apoptosis in HCC cells, we speculated the enhanced chemotherapy effects of 5-FU on HCC cells. Notably, the cell viability in Mapk10-transfected group was significantly inhibited as compared to that in the control group (Fig. 8A). Furthermore, the IC50 of HepG2 cells for 5-FU was significantly reduced after Mapk10 transfection, which was 16.253 µg/mL in the Mapk10-transfected group as compared to 251.356 µg/mL for the control group (Fig. 8B).