Upregulated PrPC by HBx enhances NF-κB signal via liquid-liquid phase separation to advance liver cancer

Abstract

Cellular prion protein (PrP^C) has been implicated in carcinogenic through the activation of various signal pathways, however, the precise mechanisms remain elusive. In vitro studies have shown that PrP^C is prone to undergo liquid-liquid phase separation (LLPS). However, it remains unknown whether PrP^C contributes to LLPS-inducing cancer development. Herein, we observed an upregulation of PrP^C expression in hepatitis B virus-positive hepatocellular carcinoma (HCC). Subsequent investigation revealed that HBx of HBV enhances PrP^C expression in a dose-dependent manner by binding to CREB1. Furthermore, we demonstrated that PrP^C undergoes LLPS and recruits TRAF2/6, TAB2/3, and TAK1 protein, thereby activating the NF-κB signaling pathway and promoting tumor progression. Notably, although unable to undergo LLPS itself, the α3 helix of PrP^C is essential for its activation of the NF-κB signaling pathway during the LLPS process. Further analysis unveiled that disulfide bond formation within the C-terminal domain of PrP^C is necessary for its LLPS and subsequent activation of the NF-κB signaling pathway. Additionally, our findings indicate that NF-κB activation by PrP^C condensates leads to increased IL-8 expression which further promotes tumor development.

Fig. 1. PrP^C is more highly expressed in HBV-positive liver cancer tissues than in normal liver tissues.

A TCGA and GTEx databases were used to analyze PrP^C mRNA expression in liver cancer and normal liver tissues. The line shows the average value. Data were analyzed using Wilcoxon two-tailed t-tests. The data used in this study are standardized Transcripts Per Kilobase of exon model per Million mapped reads (TPM) data. B Overall survival rate of LIHC patients with high or low levels of PRNP mRNA in liver cancer tissues in the GEPIA website. Group cutoff: high=50%, low = 50%, high = 182, low = 182. C TCGA and GTEx databases were used to analyze PrP^C mRNA expression in HBV-positive liver cancer and normal liver tissues. The line shows the average value. Data were analyzed using Wilcoxon two-tailed t-tests. The data used in this study are standardized TPM data. D Expression levels of PrP^C in HepG2 and HepG2 2.15 cells were detected by qPCR and western blotting. E The expression of PrP^C in HepAD38 cells was detected by qPCR and western blotting. Tetracycline was used to inhibit HBV production by HepAD38 cells. F qPCR and western blotting were used to measure the expression of PrP^C in HepG2-NTCP cells infected with HBV. MOI = 500. G qPCR and western blotting were used to measure changes in PrP^C expression in HepG2 and Huh7 cells transfected with pHBV1.3 plasmids. + represents 1 µg plasmid, and ++ represents 2 µg plasmid. H The mRNA levels of PrP^C were detected after transfection of empty vector, pXJ40-HA-HBx, pXJ40-HA-HBS, pXJ40-HA-HBC, and pXJ40-HA-HBP in HepG2 cells. I After transfection with pcDNA3.0, pcDNA3.0-HBV1.2, or pcDNA3.0-HBV1.2-X^-, the mRNA and protein expression levels of PrP^C in HepG2 cells were detected. J Luciferase activity of different PRNP promoter constructs in 293T cells transfected with HBx or empty vector (left). Luciferase activity of PRNP (-500-0 bp) promoter constructs in 293T cells transfected with transcription factors CREB1, YY1, XBP1, CEBPB, GR-α, TFID, or empty vector (right). K Huh7 cells transfected with Flag-CREB1 and HA-HBx were subjected to ChIP assay with three sets of primers in the PRNP promoter region (-500-0 bp). L Western blotting was used to measure the protein level of PrP^C in HepG2 2.15 cells transfected with pXJ40-flag-CREB1 or empty vector, CREB1 siRNAs, or siNC. M HepG2 cells were co-transfected with the plasmid expressing HBx and/or CREB1 siRNAs or siNC, and the PrP^C protein level was analyzed. Data are means ± SEM. Student’s t-tests were used to determine group differences. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 2. PrP^C promotes the proliferation and migration of hepatoma cells by activating the NF-κB signaling pathway.

A EdU assays (left) and transwell experiments (right) were performed to screen for the signaling pathways required for the effects of PrP^C on HepG2 2.15 cells. Signaling pathway inhibitors (100 μM PD98059 for MAPK/ERK, 10 μM MK2206 for AKT, 10 μM SC-514 for NF-κB, 10 μM SP600125 for JNK, 1 μM rapamycin for mTOR, 10 μM XAV-939 for Wnt and control agent DMSO) were added to cell culture medium 24 h after transfection. B Immunoblotting assays were used to detect marker proteins for cell proliferation, migration, and NF-κB pathway activation after knockdown or overexpression of PrP^C in HepG2 2.15 cells. C Nuclear localization of the p65 protein was detected in HepG2 2.15 cells after overexpression or knockdown of PrP^C. D HEK293T cells were transfected with NF-κB-luciferase (Luc) reporter along with empty vector (EV) or plasmids expressing PrP^C followed by SeV treatment for 8 h. Lysates were collected 36 h post-transfection, and luciferase activities were measured. E EdU assays were performed after PrP^C was overexpressed in HepG2 2.15 cells followed by treatment with the inhibitors SC-514 (100 μM) (left) and TPCA (100 μM) (right). F, G Transwell assays were performed to detect the migration (F) and invasion (G) of HepG2 2.15 cells after overexpression of PrP^C, and cells were treated with the inhibitors SC-514 (100 μM) (left) and TPCA (100 μM) (right). H Western blotting assays were performed to detect marker proteins for cell proliferation, migration, and NF-κB pathway activation in HepG2 2.15 cells after overexpression of PrP^C and treatment with SC-514 (100 μM). Data are means ± SEM. Student’s t-tests were used to determine group differences. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3. PrP^C activates the NF-κB pathway at the TAB2/TAB3 and its upstream level.

A Schematic diagram of NF-κB signaling pathway. C HEK293T cells were transiently transfected with NF-κB luciferase reporter, pRL-TK, or empty vector, and increasing amounts of PrP^C together with plasmids expressing MYD88, TRAF2, TRAF6, TAK1, TAB1, TAB2, TAB3, IKKβ, or p65. The cell lysate was collected 36 h after transfection, and luciferase activity was detected. D Schematic illustration of PrP^C domain organization. B, E, F HEK293T cells were transiently transfected with plasmids expressing HA-PrP^23-231 (B), HA-PrP^23-199 (E), and HA-PrP^125-231 (F) proteins together with plasmids expressing Flag-MYD88, Flag-TRAF2, Flag-TRAF6, Flag-TAK1 and Flag-TAB1, Flag-TAB2, Flag-TAB3, Flag-IKKα, Flag-P65, or Flag-empty vector, and cells were harvested 36 h later. Whole-cell lysates were subjected to immunoprecipitation with anti-Flag beads and analyzed by western blotting with the indicated antibodies. *p < 0.05; G HEK293T cells were transfected with NF-κB luciferase reporter, pRL-TK, and pXJ40-HA, pXJ40-HA-PrP^C, pXJ40-HA-PrP^23-124, pXJ40-HA-PrP^125-231, pXJ40-HA-PrP^23-199, pXJ40-HA-PrP^23-172, or pXJ40-HA-PrP^23-154 plasmids. Cells were collected 36 h after transfection, and SeV was added to the medium 8 h before sample collection (left). MYD88 was used instead of SeV to activate the NF-κB pathway (right). Luciferase activity was measured after cell lysis. Data are means ± SEM. Student’s t-tests were used to determine group differences. *p < 0.05; **p < 0.01; ***p < 0.001, ns: no significance.

Fig. 4. PrP^C forms biomolecular condensates to activate the NF-κB signaling pathway through LLPS in vivo.

A Immunofluorescence staining of endogenous PrP^C in HepG2 and HepG2 2.15 cells. Quantitative analysis of the puncta shown to the right. B Immunofluorescence staining of endogenous PrP^C in Huh7 cells and Huh7 cells transfected with pHBV1.3 plasmids. Quantitative analysis of the puncta shown to the right. C Representative live-cell images of HepG2 2.15 cells stably expressing EGFP or EGFP-PrP^C protein. D HEK293T cells were transfected with plasmids expressing HA-PrP^23-231, flag-empty vector, or Flag-PrP^23-231 and treated with SeV for 8 h followed by 2% 1,6-hexanediol 1 h before harvest. Whole-cell lysates were immunoprecipitated with anti-Flag beads and analyzed by western blotting with corresponding antibodies. E Plasmids expressing PrP^C or empty vectors were transfected into HepG2 2.15 cells, and 2% 1,6-hexanediol was added 1 h before cells were harvested 48 h after transfection. Whole-cell lysates were subjected to native PAGE. Upper panel: short exposure; lower panel: long exposure for left two lanes. F Fusion analysis of EGFP-PrP^C condensates in HepG2 2.15 cells. G FRAP assay of EGFP-PrP^C condensates in HepG2 2.15 cells (left). Quantification of fluorescence recovery (right). H Time-lapse imaging of HepG2 2.15 cells stably expressing EGFP-PrP^C protein after 2% 1,6-hexanediol treatment. Quantitative analysis of the puncta shown to the right. I HEK293T cells were transfected with NF-κB luciferase reporter, pRL-TK, or empty vector, and increasing amounts of PrP^C together with plasmids expressing MYD88, TRAF2, TRAF6, TAB2, or TAB3 or stimulated with SeV for 8 h. Cells were collected 36 h after transfection, and 2% 1,6-hexanediol was added 1 h before sample collection. All scale bars are indicated in the figures. Data are means ± SEM. Student’s t-tests were used to determine group differences. *p < 0.05; **p < 0.01, ***p < 0.001, ns: no significance.

Fig. 5. PrP^C condensates activate the NF-κB signaling pathway by recruiting TRAF2/6, TAB2/3, and TAK1 complexes.

A Immunofluorescence was performed to stain endogenous PrP^C, TRAF2, TAB2, or TAK1 proteins in HepG2 2.15 cells to determine the co-localization between PrP^C and TRAF2/TAB2/TAK1 using confocal microscopy. Line profile of fluorescence (right). B Immunofluorescence was performed to stain endogenous PrP^C, TRAF2, TAB2, or TAK1 proteins in Huh7 cells transfected with pHBV1.3 plasmids to determine the co-localization between PrP^C and TRAF2/TAB2/TAK1 using confocal microscopy. Line profile of fluorescence (right). C Immunofluorescence staining of PrP^C (red), TAB2 (orange), TAK1(green), and TRAF2 (pink) in liver sections from a liver cancer patient. D HEK293T cells were transfected with plasmids expressing HA-TAB2, Flag-TAK1, and PrP^C and their corresponding empty plasmids. Cells were collected 36 h after transfection, and 2% 1,6-hexanediol was added 1 h before cell collection. Whole-cell lysates were immunoprecipitated with anti-Flag beads and analyzed by western blotting with corresponding antibodies. E HepG2 2.15 cells were transfected with pEF-flag and pEF-flag-PrP^C or pLKO.1-shNC and pLKO.1-shPRNP plasmids. Cells were collected 48 h after transfection, and 2% 1,6-hexanediol was added to the medium 1 h before sample collection. F Representative images showing the distribution of TAB2, TAK1, and TRAF2 in PrP^23-231 droplets in vitro. Line profile of fluorescence (down). All scale bars are indicated in the figures.

Fig. 6. The globular domain of PrP^C forms condensates and recruits TRAF2/6, TAB2/3, and TAK1 complexes to activate the NF-κB signaling pathway.

A Fusion analysis of EGFP-PrP^125-231 condensates in HepG2 2.15 cells. B FRAP assay of EGFP-PrP^125-231 condensates in HepG2 2.15 cells. The quantification of fluorescence intensity is shown below. C HepG2 2.15 cells were transfected with pHAGE-EGFP-PrP^125-231 together with plasmids expressing Flag-TRAF2, Flag-TRAF6, Flag-TAB2, Flag-TAB3, or Flag-TAK1. Immunofluorescence staining for TRAF2, TRAF6, TAB2, TAB3, and TAK1 proteins. D HepG2 2.15 cells were transfected with pHAGE-EGFP-PrP^125-231, pHAGE-mcherry-TAK1, pHAGE-EBFP-TRAF2, or pHAGE-EBFP-TAB2 plasmids to observe co-localization. E HepG2 2.15 cells were transfected with pHAGE-EGFP-PrP^125-231, pHAGE-EBFP-TRAF2, and HA-Ub-K63 plasmids. After immunofluorescence staining of HA-Ub-K63, co-localization was observed. F HEK293T cells were transfected with plasmids expressing HA-TAB2, Flag-TAK1, PrP^C, PrP^23-124, PrP^125-231, and corresponding empty plasmids and collected 36 h later. Whole-cell lysates were immunoprecipitated with anti-FLAG beads and analyzed by western blotting with corresponding antibodies. G HepG2 2.15 cells were transfected with plasmids expressing PrP^C, PrP^23-124, PrP^125-231, or empty vector and collected 48 h later. Key signaling molecules of the NF-κB pathway in cells were analyzed. H Representative images showing the distribution of TAB2, TAK1, and TRAF2 in PrP^125-231 droplets in vitro. All scale bars are indicated in the figures.

Fig. 7. PrP^125-199 and PrP^200-231 do not undergo LLPS.

A Schematic diagram of EGFP-PrP^125-231 truncation mutants. B Representative live-cell images of HepG2 2.15 cells stably expressing EGFP, EGFP-PrP^125-199, and EGFP-PrP^200-231. C FRAP assay of EGFP-PrP^125-199 condensates in HepG2 2.15 cells. Quantification of fluorescence intensity is shown above. D Representative images showing the turbidity of 20 μM EGFP, EGFP-PrP^125-231, EGFP-PrP^125-199, and EGFP-PrP^200-231 solution with or without 25% PEG8000. The indicated solution was observed under confocal microscopy. E The increasing concentration of EGFP-PrP^125-199 and EGFP-PrP^200-231 solution supplemented with 25% PEG8000 was observed under confocal microscopy. F HepG2 2.15 cells were transfected with EGFP-PrP^125-231, EGFP-PrP^125-199, EGFP-PrP^200-231, or empty vector and collected 48 h later. Key signaling molecules of the NF-κB pathway in cells were analyzed. All scale bars are indicated in the figures.

Fig. 8. The disulfide bond of PrP^C is required for the LLPS of its C-terminal domain and its activation of the NF-κB signaling pathway.

A Representative live-cell images of HepG2 2.15 cells transfected with plasmids expressing EGFP, EGFP-PrP^125-231, EGFP-PrP^125-231-C179A or EGFP-PrP^125-231-C214A. B 20 μM EGFP, EGFP-PrP^125-231, EGFP-PrP^125-231-C179A or EGFP-PrP^125-231-C214A solution supplemented with 25% PEG8000 were observed under confocal microscopy. C HEK293T cells were transiently transfected with plasmids expressing HA-PrP^125-231–C214A proteins together with plasmids expressing Flag-TRAF2, Flag-TRAF6, Flag-TAK1, Flag-TAB2, Flag-TAB3, or Flag-empty vector, and cells were harvested 36 h later. Whole-cell lysates were subjected to immunoprecipitation with anti-Flag beads and analyzed by western blotting with the indicated antibodies. D HepG2 2.15 cells were transfected with pHAGE-EGFP-PrP^125-231-C214A together with plasmids expressing Flag-TRAF2, Flag-TRAF6, Flag-TAB2, Flag-TAB3, or Flag-TAK1. Immunofluorescence staining for TRAF2, TRAF6, TAB2, TAB3, and TAK1 proteins. E HepG2 2.15 cells were transfected with pXJ40-HA, HA-PrP^125-231, HA-PrP^125-231-C179A, or HA-PrP^125-231-C214A and collected 48 h later. Key signaling molecules of the NF-κB pathway in cells were analyzed. F Transwell experiments and G EdU assays were performed after the HepG2 2.15 cells were transfected with pXJ40-HA, HA-PrP^125-231, HA-PrP^125-231-C179A or HA-PrP^125-231-C214A. All scale bars are indicated in the figures. Data are means ± SEM. Student’s t-tests were used to determine group differences. **p < 0.01; ***p < 0.001.

Fig. 9. PrP^C promotes the proliferation and metastasis of HCC in vivo.

A Western blot validation of PrP^C knockdown efficiency in HepG2 2.15 cells. B Tumor images of nude mice (left). Tumors were removed from the skin of nude mice that were injected subcutaneously with HepG2 2.15-ctrl or HepG2 2.15-sgPRNP cells (middle). Tumor weight (right). n = 7. C Representative images (left) and quantification (right) of Ki67 expression in tumors from PrP^C knockdown and control groups. Scale bar, 20 μm. n = 7. D Tumors were removed from the skin of nude mice that were injected subcutaneously with LM3-ctrl or LM3-PrP^C cells (left). Tumor weight (right). n = 6. E Representative images (left) and quantification (right) showing Ki67 expression in tumors in the PrP^C overexpression and control groups. Magnification, 400×. Scale bar, 20 μm, n = 6. F Huh7-ctrl or Huh7-PrP^C cells were injected into nude mice via the tail vein. Representative images of tumor growth in livers from the metastasis model (left). H&E staining of liver tissues (right). Tumor cells are circled. Scale bar, 100 μm. n = 6. G Representative H&E staining images showing lung metastases in the Huh7-ctrl and Huh7-PrP^C groups. Scale bar for 15×, 1000 μm; scale bar for 200×, 100 μm, n = 6. H Immunoblotting assays were used to detect marker proteins of proliferation, migration, and NF-κB pathway activation in tumors formed by HepG2 2.15-ctrl, HepG2 2.15-sgPRNP cells (left), and LM3-ctrl, LM3-PrP^C cells (right). Data are means ± SEM. Student’s t-tests were used to determine group differences. **p < 0.01; ***p < 0.001.

Fig. 10. PrP^C promotes the expression of IL-8 by activating the NF-κB signaling pathway, thereby promoting tumor progression.

A qPCR and ELISA were performed to analyze the mRNA and protein levels of IL-8 in HepG2 2.15 cells transfected with plasmids expressing PrP^C followed by treatment with the NF-κB inhibitor SC-514 24 h after transfection. Cell supernatant was collected for ELISA, and cells were collected for RNA extraction 48 h after transfection. B mRNA and protein levels of IL-8 in HepG2 2.15 cells transfected with plasmids expressing PrP^C. 2% 1,6-hexanediol was added to the cell culture medium for 2 h before cells were collected. C mRNA and protein levels of IL-8 in HepG2 2.15 cells transfected with plasmids expressing PrP^C and its truncated versions for 48 h. D Correlational analysis of PRNP and IL-8 levels in LIHC patients according to the TCGA database. E Overall survival rate of LIHC patients with high or low levels of IL-8 mRNA in liver cancer tissues from the TCGA database. F Tumors removed from the skin of nude mice were injected subcutaneously with LM3-ctrl, LM3-PrP^C-shNC, or LM3-PrP^C-shIL8 cells (left). Tumor weight (right). n = 5. G Representative images showing immunohistochemical staining of Ki-67 in tumors in the LM3-ctrl group, LM3-PrP^C-shNC, and LM3-PrP^C-shIL8 groups. Fraction of Ki67-positive cells in tumors (right). Magnification: 400×. Scale bar, 20 μm. n = 5. H Huh7-ctrl, Huh7-PrP^C-shNC, and Huh7-PrP^C-shIL8 cells were injected into nude mice via the tail vein. Representative images show tumor growth in the liver in the metastasis model. I H&E staining of liver tissues. Tumor cells are circled. Scale bar, 100 μm. n = 5. J Representative images showing H&E staining of lung tissues in the metastasis model. Scale bar for 15×, 1000 μm; scale bar for 200×, 100 μm. n = 5. Data are means ± SEM. Student’s t-tests were used to determine group differences. *p < 0.05; **p < 0.01; ***p < 0.001.