HRG switches TNFR1-mediated cell survival to apoptosis in Hepatocellular Carcinoma

Background: Tumor necrosis factor receptor 1 (TNFR1) signaling plays a pleiotropic role in the development of hepatocellular carcinoma (HCC). The formation of TNFR1-complex I supports cell survival while TNFR1-complex II leads to apoptosis, and the underlying mechanisms of the transformation of these TNFR1 complexes in HCC remain poorly defined. Methods: The interaction protein of TNFR1 was identified by GST pulldown assay, immunoprecipitation and mass spectrometry. In vitro and in vivo assay were performed to explore the biological features and mechanisms underlying the regulation of TNFR1 signals by histidine-rich glycoprotein (HRG). Data from the public databases and HCC samples were utilized to analyze the expression and clinical relevance of HRG. Results: HRG directly interacted with TNFR1 and stabilized TNFR1 protein by decreasing the Lys(K)-48 ubiquitination mediated-degradation. The formation of TNFR1-complex II was prompted by HRG overexpression via upregulating Lys(K)-63 ubiquitination of TNFR1. Besides, overexpression of HRG suppressed expression of pro-survival genes by impairing the activation of NF-κB signaling in the presence of TNFR1. Moreover, downregulation of HRG was a result of feedback inhibition of NF-κB activation in HCC. In line with the pro-apoptotic switch of TNFR1 signaling after HRG induction, overexpression of HRG inhibited cell proliferation and increased apoptosis in HCC. Conclusions: Our findings illustrate a crucial role for HRG in suppressing HCC via inclining TNFR1 to a pro-apoptotic cellular phenotype. Restoring HRG expression in HCC tissues might be a promising pharmacological approach to blocking tumor progression by shifting cellular fate from cell survival to apoptosis.

immunoprecipitation, IgG or the specific antibody was incubated with the cell supernatant (lysed using NP-40 Lysis Buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors) overnight at 4 °C. The cell extracts were then immunoprecipitated with protein A/G magnetic beads (Bbimake, China) according to the manufacturer's instruction.

Immunofluorescence assay
Huh7 and SMMC-7721 cells were seeded on coverslips for the indicated transfections.
Following incubation for 24-48 h, the cells on the coverslips were washed with phosphatebuffered saline (PBS) three times and fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.25% Triton for 5 min. Thereafter, the cells were incubated with anti-pP65 (#3033, CST, USA) antibodies at 4 °C overnight. Following washes, the cells were incubated with rhodamine-conjugated goat antibodies against rabbit IgG (Abcam, Cambridge, UK) at 37 °C for 1 h. The nuclei were stained with DAPI (Abcam, Cambridge, UK) and the cell membranes were stained with Dil (#C1036, Beyotime, Shanghai, China) at 37 °C for 20 min.
The images were captured with a BX51 or BX63 microscope (Olympus, Japan).

Dual-luciferase reporter assay
SMMC-7721 cells stably overexpressing HRG and the corresponding controls were cultured in 24-well plates and co-transfected with a NF-κB luciferase reporter. At 48 h posttransfection, luciferase activity was measured using the Dual-Luciferase Assay Kit (Promega, Madison, WI) according to the manufacturer's instructions.

Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from HCC cells or human tissues using TRIzol ® reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Tokyo, Japan) was used for first-strand cDNA synthesis.
Real-time PCR was performed using SYBR ® Green PCR kit (TaKaRa, Tokyo, Japan). The primers sequences used for the amplification are shown in Table S4.

5-ethynyl-20-deoxyuridine (EdU) assay
The HCC cell lines transfected with different vectors were seeded into 96-well plates and cultured overnight. Briefly, cells were incubated with 50 μM EdU for 2 h. After EdU-DNA incorporation, the cells were fixed and stained using EdU Cell Proliferation Assay Kit (Ribobio, Wuhan, China) according to the manufacturer's instructions.

Flow-cytometry assay
HRG-overexpressing HCC cells and the corresponding controls were stimulated with different doses of 5-fluorouracil. After 48 h, the cells were digested with EDTA-free trypsin and resuspended to generate a single-cell suspension. Apoptotic cells were evaluated using Annexin-V-APC and 7-AAD Apoptosis Detection Kits (KeyGen BioTECH, Suzhou, China).
The cells were then analyzed with a FACScan flow cytometer using the FlowJo software (Tree Star Inc., Ashland, OR, USA).

Gene set enrichment analysis (GSEA)
GSEA was used to search gene signatures that correlated with HRG expression in the TCGA dataset as previously described [25]. Briefly, patients were split into two groups according to the HRG mRNA expression in the TCGA cohort. The GSEA software (GSEA v. 2.0, http://www.broadinstitute.org/gsea) was utilized to analyze, annotate, and interpret enrichment results.

Figure S7. Correlation between HRG or TNFR1 expression and prognosis in HCC. (A-B)
Results of GSEA were plotted to visualize the correlation between the expression of HRG and gene signatures of liver cancer survival and recurrence in the TCGA cohort. (C-D) Kaplan-Meier analysis of overall survival in TCGA and GSE14520 cohorts based on TNFRSF1A expression status. X-tile software was used to generate optimal cut-off values and separate patients into high and low TNFRSF1A expression groups.