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. 2019 Nov 26;38(1):474.
doi: 10.1186/s13046-019-1442-2.

DNMT3b/OCT4 expression confers sorafenib resistance and poor prognosis of hepatocellular carcinoma through IL-6/STAT3 regulation

Ssu-Chuan Lai  1   2 , Yu-Ting Su  1   2   3 , Ching-Chi Chi  4   5 , Yung-Che Kuo  1   2   3 , Kam-Fai Lee  6 , Yu-Chih Wu  3   7 , Pei-Chi Lan  3 , Muh-Hwa Yang  8   9 , Te-Sheng Chang  10   11 , Yen-Hua Huang  12   13   14   15   16   17   18
Affiliations

DNMT3b/OCT4 expression confers sorafenib resistance and poor prognosis of hepatocellular carcinoma through IL-6/STAT3 regulation

Ssu-Chuan Lai et al. J Exp Clin Cancer Res. .

Erratum in

Abstract

Background: The inflammatory cytokine interleukin-6 (IL-6) is critical for the expression of octamer-binding transcription factor 4 (OCT4), which is highly associated with early tumor recurrence and poor prognosis of hepatocellular carcinomas (HCC). DNA methyltransferase (DNMT) family is closely linked with OCT4 expression and drug resistance. However, the underlying mechanism regarding the interplay between DNMTs and IL-6-induced OCT4 expression and the sorafenib resistance of HCC remains largely unclear.

Methods: HCC tissue samples were used to examine the association between DNMTs/OCT4 expression levels and clinical prognosis. Serum levels of IL-6 were detected using ELISA assays (n = 144). Gain- and loss-of-function experiments were performed in cell lines and mouse xenograft models to determine the underlying mechanism in vitro and in vivo.

Results: We demonstrate that levels of DNA methyltransferase 3 beta (DNMT3b) are significantly correlated with the OCT4 levels in HCC tissues (n = 144), and the OCT4 expression levels are positively associated with the serum IL-6 levels. Higher levels of IL-6, DNMT3b, or OCT4 predicted early HCC recurrence and poor prognosis. We show that IL-6/STAT3 activation increases DNMT3b/1 and OCT4 in HCC. Activated phospho-STAT3 (STAT-Y640F) significantly increased DNMT3b/OCT4, while dominant negative phospho-STAT3 (STAT-Y705F) was suppressive. Inhibiting DNMT3b with RNA interference or nanaomycin A (a selective DNMT3b inhibitor) effectively suppressed the IL-6 or STAT-Y640F-induced increase of DNMT3b-OCT4 and ALDH activity in vitro and in vivo. The fact that OCT4 regulates the DNMT1 expressions were further demonstrated either by OCT4 forced expression or DNMT1 silence. Additionally, the DNMT3b silencing reduced the OCT4 expression in sorafenib-resistant Hep3B cells with or without IL-6 treatment. Notably, targeting DNMT3b with nanaomycin A significantly increased the cell sensitivity to sorafenib, with a synergistic combination index (CI) in sorafenib-resistant Hep3B cells.

Conclusions: The DNMT3b plays a critical role in the IL-6-mediated OCT4 expression and the drug sensitivity of sorafenib-resistant HCC. The p-STAT3 activation increases the DNMT3b/OCT4 which confers the tumor early recurrence and poor prognosis of HCC patients. Findings from this study highlight the significance of IL-6-DNMT3b-mediated OCT4 expressions in future therapeutic target for patients expressing cancer stemness-related properties or sorafenib resistance in HCC.

Keywords: DNMT3b; Drug resistance; Hepatocellular carcinoma; Interleukin-6; OCT4; Phospho-STAT3.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Correlation between serum IL-6 and tissue DNMT3b/OCT4 with the patient prognosis of human HCC. The overall survival (OS) (a) and early tumor recurrence (within 24 months) (b) of patients after HCC resection based on high or low serum IL-6 level by Kaplan-Meier analysis (n = 144, cutoff for high IL-6 concentration was 150 pg/mL). After normalization with the corresponding peritumor (PT) tissue sample, the expression levels (either high [T/PT ≧ 2 or low [T/PT < 2]) of OCT4 were assessed. c The differences in serum levels of IL-6 between HCC patients with low OCT4 expression (T/PT < 2-fold; n = 101) and high OCT4 expression (T/PT ≥ 2-fold; n = 43) are shown. Positive correlations by Spearman analysis between expression levels of OCT4 with DNMT3b (R = 0.7253) (d) and DNMT1 (R = 0.4771) (e) in HCC tissues. n = 144. The differences in DNMT3b between HCC patients with low OCT4 expression (T/PT < 2-fold; n = 108) and high OCT4 expression (T/PT ≥ 2-fold; n = 36) (f) Statistical significance was assessed by the Mann–Whitney U test. (*P < 0.05; ***P < 0.001). g The Kaplan-Meier curves of tumor recurrence (120 months) or early recurrence (24 months) in relation to the transcriptional levels of OCT4 (n = 144), DNMT3b (n = 144) in human HCC tissue (*P < 0.05, **P < 0.01). h The OS analysis of DNMT expression in HCC using The Cancer Genome Atlas (TCGA) dataset by Kaplan-Meier analysis (n = 364). The top tertile was defined as the high DNMTs expression cohort and the remaining patients were defined as the low DNMTs expression cohort. i The expression and localization of DNMT3b, OCT4, and DNMT1 in HCC tissues by immuno-histochemical staining. (Bar, 100 μm)
Fig. 2
Fig. 2
IL-6 activates the expression of DNMTs and OCT4 in vitro and in vivo. a The effect of IL-6 (50 ng/mL) on mRNA expression of DNMTs and OCT4 in HBV+HBsAg+ HCCs (Hep3B and HepG2.2.15) and HBV−HBsAg− HCCs (HepG2) under different incubation time assessed by real-time qPCR. b The effect of IL-6 (50 ng/mL) on the protein expression of DNMTs and OCT4 in HBV+HBsAg+ HCCs (Hep3B and HepG2.2.15) and HBV−HBsAg− HCCs (HepG2 and Huh7) by western blotting. The qualitative data is shown in (c). Three individual experiments were carried out for each experimental condition. *P < 0.05, **P < 0.01, ***P < 0.0001, student t test. d Xenografts of 5 × 106 Hep3B cells in athymic nude mice with IL-6 (200 ng/mouse/3 d) treatment (n = 6). Immuno-histochemical staining for p-STAT3, DNMT3b, OCT4, DNMT1, DNMT3a, and the control IgG are shown (Bar, 100 μm)
Fig. 3
Fig. 3
p-STAT3 activation enhances the expression of DNMTs/OCT4 and increases ALDH activity in Hep3B cells. The expression levels of DNMTs and OCT4 in Hep3B overexpressed with STAT3-WT, STAT3-Y640F (constitutively phosphorylated) or STAT3-Y705F (dominant negative). a Western blot analysis. b The quantitative data of (a). *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, student t test. c ALDH activity in Hep3B cells transfected with STAT3-WT, STAT3-Y640F or STAT3-Y705F. d The quantitative data of (c). *P < 0.05, student t test. ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; SSC, side scatter. e The protein levels of DNMT3b and OCT4 in STAT3-Y640F-Hep3B cells with or without RNA silencing of DNMT3b or DNMT1. shCtrl, control vector; shDNMT3b, DNMT3b silencing plasmid; and shDNMT1, DNMT1 silencing plasmid. e Western blot analysis. f The quantitative data of (e). *P < 0.05, **P < 0.01, student t test
Fig. 4
Fig. 4
DNMT3b silencing reduces IL-6-induced OCT4 and forced OCT4 expression increases the DNMT1 in Hep3B cells. a Effect of DNMT3b silencing on the IL-6-induced OCT4 expression in Hep3B cells. b OCT4 and DNMT1 protein levels of the shCtrl.- or shDNMT3b Hep3B cells. c Effect of forced OCT4 expression on the protein levels of DNMT1, DNMT3a, and DNMT3b in Hep3B cells. d DNMT3b and OCT4 protein levels of the shCtrl.- or shDNMT1 Hep3B cells. Western blotting analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, student t test
Fig. 5
Fig. 5
IL-6 increases OCT4 expression through DNMT3b regulation in vitro and in vivo. a Effect of nanaomycin A (a DNMT3b inhibitor) on protein expression of DNMT3b/1, OCT4, and N-cadherin in Hep3B cells. b The effect of nanaomycin A (0, 1, 5, 10, 20 μM) on the migration ability of Hep3B cells. Transwell assay. Bar = 100 um. c Effect of nanaomycin A on IL-6-induced protein expression of DNMT3b/1 and OCT4 in Hep3B cells. d The time course of IL-6/nanaomycin A-treated Hep3B animal models. n = 6 for each group. e Immunohistochemical analysis of the protein expressions of p-STAT3, STAT3, DNMT3b, OCT4, and DNMT1 following PBS, IL-6, and IL-6 plus nanaomycin A treatment (0, 2, 20 μM/mouse/2 days). Bar, 100 μm. f The quantitative data of (a). *P < 0.05, **P < 0.01, ***P < 0.001, student t test
Fig. 6
Fig. 6
Combination of nanaomycin A increases the sorafenib susceptibility in Hep3B sorafenib resistant HCC cells. (a) The cell viability of Sorafenib-naïve Hep3B cells or sorafenib-resistant Hep3B cells under Sorafenib treatment (0, 1, 5, 10, 15 20 μM) (b) The mRNA levels of IL-6R, stemness-related genes, and DNMTs in Hep3B naïve/resistant cells were shown. Real-time Q-PCR assay. *P < 0.05, **P < 0.01, ***P < 0.001, student t test. (c, d) The cell viability of Hep3B naïve/resistant cells under the treatment of Sorafenib (1, 5, 10, 15 20 μM) with or without nanaomycin A (2, 5, 10 μM) for 48 h. The WST assay. *P < 0.05, **P < 0.01, ***P < 0.001, student t test. e The calculated combination index (CI) values of the (c, d) were shown. The meaning of CI was interpreted as: CI > 1, antagonistic effect; CI = 1, additive effect; and CI <  1, synergistic effect
Fig. 7
Fig. 7
IL-6 increases the expression of OCT4 through DNMT3b and IGF-1R activation in human HCC. a Model of how IL-6 increases the expression of OCT4 through p-STAT3-DNMT3b-OCT4-DNMT1 activation in human HCC. b The combination use of nanaomycin and sorafenib synergistically suppresses the cell proliferation of sorafenib resistant HCC cells
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