Honokiol Prevents Non-Alcoholic Steatohepatitis-Induced Liver Cancer via EGFR Degradation through the Glucocorticoid Receptor—MIG6 Axis

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  Figure 1. Honokiol (HNK) treatment attenuates hepatocellular carcinoma (HCC) development in major urinary protein (MUP)-urokinase type plasminogen activator (uPA) transgenic mice. (A) Protocol for honokiol treatment in MUP-uPA transgenic mice fed a high-fat diet (HFD). Then, six week-old male mice fed an HFD for 26 weeks were treated with vehicle control or HNK (10 mg/kg, intraperitoneally (three times/week) for another 8 weeks (32–40 weeks of age). Tumor development was analyzed at 40 weeks. For the non-alcoholic steatohepatitis (NASH) analysis, 6-week-old male mice fed an HFD for 6 weeks were treated with vehicle or HNK for 8 weeks (12–20 weeks of age). During honokiol treatment, the HFD feeding regimen was continued. (B) Gross morphology of livers with HCC and typical HCC histology in mice of the MUP-HFD model. (C) Maximal tumor size and tumor numbers in MUP-HFD mice treated with vehicle or honokiol. Tumor development was analyzed 2–4 days after the final honokiol injection. Results are presented as the median with interquartile ranges. (D) Liver sections of MUP-HFD mice were analyzed at 20 weeks of age after treatment with vehicle or honokiol for the last 8 weeks of NASH development.…

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  Figure 2. Epidermal growth factor receptor (EGFR) signaling is upregulated in HFD-fed MUP-uPA mouse liver. (A) Hierarchical clustering results of differentially expressed genes between HFD-fed MUP-uPA (MUP) and HFD-fed wild-type B6 (WT) mouse livers (n = 3 per group). The list of genes analyzed here is shown in Table S1. (B) A total of 21 genes, including Egfr and Fkbp5, were upregulated in MUP-uPA mice, and suppressed by HNK-treatment.

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  Figure 3. Honokiol (HNK) treatment suppresses epidermal growth factor receptor (EGFR) signaling in the non-alcoholic steatohepatitis (NASH) liver. (A) Relative expression of Egfr, FK506 binding protein 5 (Fkbp5), and ERBB receptor feedback inhibitor 1 (ERRFI1) mRNAs in non-tumor livers from major urinary protein (MUP)-urokinase type plasminogen activator (uPA) transgenic mice receiving vehicle control (MUP) or HNK and their wild-type (WT) littermates receiving vehicle control (n = 5 per group). All mice were fed a high-fat diet (HFD) from 6 weeks of age until sacrifice at 40 weeks of age. Results are presented as the mean ± SD (# p < 0.01). (B) Immunoblot analysis of the non-tumor liver extracts from WT, MUP, and HNK mice. Protein expression and phosphorylation of EGFR signaling-related molecules, and FKBP5 are presented (n = 3 per group). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Relative cell viability and proliferation, and (D) relative ERRFI1 mRNA expression in Hep3B cells 24, 36, and 48 h after incubation with 0, 10, 20, or 30 μM HNK. All graphs represent the mean ± SD (* p < 0.05, # p < 0.01 vs. HNK0 condition at the same time course). (E)…

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  Figure 4. Mitogen-inducible gene 6 (MIG6) knockout (KO) abrogates the inhibitory effects of honokiol (HNK) on hepatocellular carcinoma (HCC) cell proliferation.(A) Viability and proliferation of parental and CRISPR MIG6 KO Hep3B cells 48 and 72 h after incubation with 0, 20, or 30 μM HNK. (B) Relative mRNA expression of ERBB receptor feedback inhibitor 1 (ERRFI1) in HCC cells treated with 0, 10, 20, or 30 μM HNK (WT, wild-type Hep3B cells; KO, CRISPR MIG6-knockout clone cells). (C) Immunoblot analysis of parental and CRISPR MIG6 KO Hep3B cells 24 h after incubation with the indicated concentrations of HNK. Protein expression and phosphorylation of EGFR signaling-related molecules are presented with β-actin as a loading control. All graphs represent the mean ± SD (* p < 0.05, # p < 0.01 vs. the same cells without HNK treatment (HNK0, A) or vs. WT cells (HNK0, B)).

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  Figure 5. Honokiol (HNK) induces glucocorticoid receptor (GR) nuclear translocation, leading to mitogen-inducible gene 6 (MIG6) induction in Hep3B cells. (A) Subcellular localization of GR in Hep3B cells 2 h after incubation with the vehicle, 20 μM HNK, or 100 nM dexamethasone (DEX) was examined via immunofluorescence staining. 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. (B) Immunoblot analysis of nuclear (-N) and cytoplasmic (-C) extracts from Hep3B cells 9 h after incubation with 0, 10, or 20 μM HNK. Protein expression of GR, epidermal growth factor receptor (EGFR), and MIG6 was examined. Lamin B1 (nuclear) and GAPDH (cytoplasmic) were used as loading controls. (C) Relative expression of ERRFI1 mRNA in Hep3B cells treated with the indicated concentrations of HNK. All graphs represent the mean ± SD (* p < 0.05, # p < 0.01 vs. HNK0 condition at the same time course).

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  Figure 6. Glucocorticoid receptor (GR) activation and ERBB receptor feedback inhibitor 1 (ERRFI1) expression are inversely correlated with epidermal growth factor receptor (EGFR) expression in human hepatocellular carcinoma (HCC). (A,B) Representative images of EGFR (A) and glucocorticoid receptor (GR; B) immunohistochemistry of non-tumor HCC adjacent tissue (N: left panels) and HCC (T: right panels). All samples were obtained via surgical resection in patients diagnosed with HCC. (C,D) Correlation between ERRFI1 mRNA expression and positivity for EGFR (C) or GR (D) in non-tumor (N) and tumor (T) tissues. Each box plot depicts the median and quartiles. Whiskers indicate the furthest point within 1.5 × the interquartile range (the third quartile minus the first quartile) from the box.

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  Figure 7. ERBB receptor feedback inhibitor 1 (ERRFI1) expression decreases in parallel with non-alcoholic steatohepatitis (NASH) progression.

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  Figure 8. Proposed mechanism of glucocorticoid receptor (GR)–mitogen-inducible gene 6 (MIG6)-mediated epidermal growth factor receptor (EGFR) degradation and suppression by honokiol. (A) EGFR signaling plays a pivotal role in liver tumorigenesis by activating RAS/RAF/ERK and mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) signaling. FK506 binding protein 5 (FKBP5) and heat shock protein (HSP) cooperate to stabilize GR and inhibit its nuclear translocation in EGFR-expressing hepatocellular carcinoma cells. (B) Honokiol may cause GR to dissociate from the FKBP5/HSP complex. Once GR becomes free, it translocates to the nucleus and induces MIG6/ERRFI1 transcription to eventually cause the lysosomal degradation of EGFR. As a result, EGFR signaling and its downstream targets RAS/RAF/ERK and mTORC1 are suppressed.