SYVN1 ubiquitinates FoxO1 to induce β-catenin nuclear translocation, PD-L1-mediated metastasis, and immune evasion of hepatocellular carcinoma

Abstract

Background: A high incidence of hepatocellular carcinoma (HCC), the most frequently diagnosed form of liver cancer, is observed in Africa and Asia. SYVN1 is upregulated in HCC; however, the biological roles of SYVN1 in immune evasion remain unclear.

Methods: RT-qPCR and western blot were employed to detect the expression levels of SYVN1 and the key molecules in HCC cells and tissues. Flow cytometry was used to determine the proportion of T cells, and an ELISA assay was used to determine the amount of IFN-γ secreted. Cell viability was monitored by CCK-8 and colony formation assays. The metastatic properties of HCC cells were detected by Transwell assays. Bioinformatics analysis, ChIP, and luciferase assays were used to study the transcriptional regulation of PD-L1. Co-IP was used to detect direct interaction between SYVN1 and FoxO1, as well as the ubiquitination of FoxO1. The in vitro findings were validated in xenograft and lung metastasis models.

Results: In HCC cells and tissues, SYVN1 was upregulated while FoxO1 was downregulated. SYVN1 knockdown or FoxO1 overexpression reduced PD-L1 expression, and inhibited immune evasion, cell growth, and metastasis in HCC cells. Mechanistically, FoxO1 regulated PD-L1 transcription in a β-catenin-independent or -dependent manner. Functional studies further showed that SYVN1 promoted immune evasion, cell proliferation, migration and invasion via facilitating ubiquitin-proteasome-dependent degradation of FoxO1. In vivo investigations showed that silencing of SYVN1 inhibited immune evasion and metastasis of HCC cells, possible via the FoxO1/PD-L1 axis.

Conclusion: SYVN1 regulates FoxO1 ubiquitination to stimulate β-catenin nuclear translocation and promotes PD-L1-mediated metastasis and immune evasion in HCC.

Keywords: E3 ubiquitin ligase SYVN1; FoxO1; Hepatocellular carcinoma; PD-L1; β-catenin.

Fig. 1

SYVN1 is upregulated, and FoxO1 is downregulated in HCC tissues and cells. (A) Data analyses based on TCGA database. (B) The protein levels of SYVN1 and FoxO1 in HCC tissues (n = 10) were detected by western blot. (C) The mRNA levels of SYVN1 and FoxO1 in HCC tissues (n = 30) were detected by RT-qPCR. (D) The protein levels of SYVN1 and FoxO1 in HCC cells were detected by western blot. **, P < 0.01, ***, P < 0.001

Fig. 2

Knockdown of SYVN1 decreases PD-L1 expression, and inhibits immune escape in HCC cells. (A) The protein levels of SYVN1 and PD-L1 in Hep3B and MHCC97H cells were detected by western blot. (B) Immunofluorescence staining of PD-L1 in Hep3B and MHCC97H cells were detected. Red, PD-L1; Blue, DAPI. Scale bar, 25 μm. (C) The proportion of CD3+/CD8 + or CD3+/CD4 + T cells were detected by flow cytometry. (D) The secreted IFN-γ level was assessed by ELISA assay. *, P < 0.05, **, P < 0.01

Fig. 3

FoxO1 overexpression decreases PD-L1 level, and suppresses immune escape, cell proliferation and metastasis in HCC cells. (A) The protein levels of FoxO1 and PD-L1 in Hep3B and MHCC97H cells were detected by western blot. (B) Immunofluorescence staining of PD-L1 in Hep3B and MHCC97H cells were detected. Red, PD-L1; Blue, DAPI. Scale bar, 25 μm. (C) The proportion of CD3+/CD8 + or CD3+/CD4 + T cells were detected by flow cytometry. (D) The secreted IFN-γ level was assessed by ELISA assay. (E) Cell proliferation was monitored by CCK-8 assay. (F) Colony forming ability was assessed by colony formation assay. (G) Cell migration and invasion were detected by Transwell assay. *, P < 0.05, **, P < 0.01, ***, P < 0.001

Fig. 4

FoxO1 regulates PD-L1 expression in a β-catenin-independent or -dependent manner. (A) The putative binding sites between FoxO1 and PD-L1 promoter were predicted by AnimalTFDB. (B) The enrichment of FoxO1 on PD-L1 promoter was assessed by ChIP assay. (C) The relative luciferase activity was detected by dual-luciferase reporter assay. (D) The protein levels of FoxO1, β-catenin and PD-L1 were detected by western blot. (E) The protein levels of β-catenin and PD-L1 were detected by western blot. (F) The putative binding sites between β-catenin and PD-L1 promoter were predicted by AnimalTFDB. (G) The enrichment of β-catenin on PD-L1 promoter was assessed by ChIP assay. (H) The relative luciferase activity was detected by dual-luciferase reporter assay. *, P < 0.05, **, P < 0.01, ***, P < 0.001

Fig. 5

SYVN1 mediates FoxO1 degradation via ubiquitin-proteasome pathway. (A) S beads-enriched complex was detected by western blot. (B) The direct interaction between SYVN1 and FoxO1 in Hep3B and MHCC97H cells was assessed by Co-IP. Normal IgG served as a negative control. Whole cell lysates were used as an input control. (C) The protein levels of FoxO1 and SYVN1 were detected by western blot. (D) The exogenous expression of FoxO1 was detected by western blot using Myc-tagged antibody. (E-F) The protein stability of FoxO1 was monitored by western blot in the presence of CHX. (G) S beads-enriched complex was detected by western blot. S beads, streptavidin beads; WCL, whole cell lysates. *, P < 0.05, **, P < 0.01, ***, P < 0.001

Fig. 6

SYVN1 promotes immune escape via modulating FoxO1. (A) The protein levels of SYVN1, FoxO1, β-catenin and PD-L1 were detected by western blot. (B) The proportion of CD3+/CD8 + or CD3+/CD4 + T cells were detected by flow cytometry. (C) The secreted IFN-γ level was assessed by ELISA assay. *, P < 0.05, **, P < 0.01, ***, P < 0.001

Fig. 7

Silencing of SYVN1 inhibits immune escape of HCC cells, possible via FoxO1/PD-L1 axis in vivo. NOD/SCID mice were subcutaneously injected with stably transfected HCC cells to establish xenograft model. (A) Tumor regression rate was calculated. (B) Percentage of Ki67 + cells in tumor sections. (C) Photographs of xenograft tumors. (D) Quantitative analysis of tumor volume. (E) Quantitative analysis of tumor weight. Data are presented as mean ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001

Fig. 8

The working mechanism of the article. SYVN1 mediated the ubiquitin-proteasomal degradation of FoxO1, thus inducing β-catenin nuclear translocation, PD-L1-mediated metastasis and immune evasion of HCC.