Opposing regulation of the STING pathway in hepatic stellate cells by NBR1 and p62 determines the progression of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) emerges from chronic inflammation, to which activation of hepatic stellate cells (HSCs) contributes by shaping a pro-tumorigenic microenvironment. Key to this process is p62, whose inactivation leads to enhanced hepatocarcinogenesis. Here, we show that p62 activates the interferon (IFN) cascade by promoting STING ubiquitination by tripartite motif protein 32 (TRIM32) in HSCs. p62, binding neighbor of BRCA1 gene 1 (NBR1) and STING, triggers the IFN cascade by displacing NBR1, which normally prevents the interaction of TRIM32 with STING and its subsequent activation. Furthermore, NBR1 also antagonizes STING by promoting its trafficking to the endosome-lysosomal compartment for degradation independent of autophagy. Of functional relevance, NBR1 deletion completely reverts the tumor-promoting function of p62-deficient HSCs by rescuing the inhibited STING-IFN pathway, thus enhancing anti-tumor responses mediated by CD8⁺ T cells. Therefore, NBR1 emerges as a synthetic vulnerability of p62 deficiency in HSCs by promoting the STING/IFN pathway, which boosts anti-tumor CD8⁺ T cell responses to restrain HCC progression.

Keywords: CD8(+) T cells; NBR1; STING; TRIM32; hepatic stellate cells; hepatocellular carcinoma; interferon; microenvironment; p62.

Figure 1.. NBR1 inactivation impairs enhanced liver tumorigenesis driven by p62-deficiency

(A–G) DEN/HFD induced HCC model in WT (n = 5), Sqstm1^−/− (n = 5), Nbr1^−/− (n = 4), and Sqstm1^−/−Nbr1^−/− (n = 5) mice. Experimental design (A), liver images (B), liver-to-body weight ratio (C), total number of tumors (D), and number of tumors bigger than 3 mm (E). Scale bar, 1 cm. qPCR analyses of Afp of WT (n = 8), Sqstm1^−/− (n = 12), Nbr1^−/− (n = 8), and Sqstm1^−/−Nbr1^−/− (n = 10) (F). Histological characterization of tumors from WT, Sqstm1^−/−, Nbr1^−/−, and Sqstm1^−/−Nbr1^−/− mice (G). (H–J) Orthotopic co-implantation HCC model in WT (n = 5), Sqstm1^−/− (n = 5), Nbr1^−/− (n = 4), and Sqstm1^−/−Nbr1^−/− (n = 5) mice. Experimental design (H), liver images (I), and tumor volume (J). Scale bar, 1 cm. (K–M) Orthotopic co-implantation HCC model. Experimental design (K), liver images (L), and tumor volume (M) from WT mice implanted with DihXD3 tumor cells with WT (n = 5), Nbr1^−/− (n = 7), Sqstm1^−/− (n = 7) or Sqstm1^−/−Nbr1^−/− (n = 7) HSCs. Scale bar, 1 cm. (N–P) Orthotopic co-implantation HCC model in Nbr1^f/f (n = 5) and Nbr1^f/f;Gfap-Cre (n = 5) mice. Experimental design (N), liver images (O), and tumor volume (P) of Nbr1^f/f and Nbr1^f/f;Gfap-Cre mice. Scale bar, 1 cm. Results are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S1.

Figure 2.. NBR1 and p62 antagonistically regulate the interferon pathway in HSCs

(A) GSEA plot in the transcriptomic data of Nbr1^−/− as compared with WT HSCs. (B) GSEA of RNA-seq of Sqstm1^−/− as compared with WT HSCs. (C) GSEA of RNA-seq of Sqstm1^−/−Nbr1^−/− as compared with WT HSCs. (D) qPCR of IFN genes in WT, Sqstm1^−/−, Nbr1^−/− and Sqstm1^−/−Nbr1^−/− HSCs (n = 3 biological replicates). (E–G) Orthotopic co-implantation HCC model. Experimental design (E); pSTAT1 and CD8 staining (F); and quantification (G) in tumors from co-implanted with WT, Sqstm1^−/−, Nbr1^−/−, Sqstm1^−/− Nbr1^−/− HSCs. Scale bar, 50 µm. (H–J) Orthotopic co-implantation HCC model in Nbr1^f/f and Nbr1^f/f;Gfap-Cre mice. Experimental design (H). pSTAT1 and CD8 staining (I) and quantification (J). Scale bar, 100 µm (CD8), and 50 µm (pSTAT1). (K–Q) CD8⁺ cell depletion in orthotopic co-implantation HCC model. Experimental design (K); liver images (scale bar, 1 cm) (L); tumor volume (n = 7 in control; n = 9 for anti-CD8) (M); quantification of incidence of lung metastasis (n = 3) (N); and H&E staining of livers and lungs. Black dotted lines delineate tumor nodules. Scale bar, 100 µm (O); flow cytometry of CD4⁺ T cell proportion among CD45⁺ cells and the IFNγ producing cells in CD4⁺ T cells (n = 3) (P); and qPCR of Ifng, Cxcl9, Cxcl10, Cd274, and Nlrc5 (n = 4 biological replicates for control; n = 3 biological replicates for anti-CD8) (Q). Results are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S2.

Figure 3.. STING is critical for the anti-tumor activity of NBR1 deficiency in HSCs

(A and B) Immunoblot (A) and qPCR (n = 3 biological replicates) (B) in Sqstm1^−/−Nbr1^−/− HSCs transfected with siRNAs. (C–F) Immunoblots (C); quantification (D and F); and qPCRs (E) in WT, Nbr1^−/−, Sqstm1^−/−, and Sqstm1^−/−Nbr1^−/− HSCs stimulated with dsDNA for the indicated times (n = 3 biological replicates). (G–I) Orthotopic co-implantation HCC model in Nbr1^f/f (n = 5) and Nbr1^f/f;Gfap-Cre (n = 5). Experimental design (G); STING, αSMA, and DESMIN (DES) staining (H); and quantification (I). Red arrows indicate STING/αSMA or STING/DES-positive cells. Scale bar, 20 and 10 µm (inserts). (J–R) Orthotopic co-implantation of DihXD3 cells with Sqstm1^−/− shNT (n = 6), Sqstm1^−/−Nbr1^−/− shNT (n = 6), or shTmem173 (n = 5) HSCs in pre-conditioned WT mice. Experimental design (J); images of liver (scale bar, 1 cm) (K); liver-to-body weight ratio (L); tumor volume (Sqstm1^−/− shNT: n = 17, Sqstm1^−/−Nbr1^−/− shNT: n = 18, and shTmem173: n = 12 HSCs) (M); and H&E staining of livers and lungs. Black dotted lines delineate tumor nodules. Scale bar, 100 µm (N); quantification of incidence of lung metastasis (Sqstm1^−/− shNT: n = 6, Sqstm1^−/−Nbr1^−/− shNT: n = 5, and shTmem173: n = 5 HSCs) (O); and flow cytometry of IFNγ producing cells among CD8⁺ and CD4⁺ T cells in CD45⁺ cells (P) or MHCII on CD11c⁻ F4/80⁺ CD11b⁻ macrophages in CD45⁺ cells and PD-L1 on CD45⁻ cells (Q) (Sqstm1^−/− shNT: n = 6, Sqstm1^−/−Nbr1^−/− shNT: n = 5, and shTmem173: n = 3 HSCs). (R) qPCR of Ifng, Cxcl9, Cxcl10, Cd274, and Nlrc5 (n = 4 biological replicates) in implanted tumors from Figure 3K. Immunoblot experiments are representative of two independent experiments. Results are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S3.

Figure 4.. NBR1 but not p62 regulates STING protein levels by controlling its trafficking from the Golgi to the lysosome

(A and B) Immunoblots in Nbr1^−/−, Sqstm1^−/−, and WT HSCs (A) or in NBR1 OV, p62 OV, and EV HSCs (B). (C and D) Immunoblots in EV and NBR1 OV HSCs treated with BafA1 (100 nM) or MG132 (50 µM) for 12 h (C) or treated with BafA1 (20 nM) for 12 h and stimulated with dsDNA for 6 h (D). (E and F) Immunoblots in EV and NBR1 OV HSCs transduced with the indicated siRNAs. (G) Scheme showing STING trafficking after cGAMP stimulation. (H–K) Immunofluorescence staining and quantification of STING with GM130 (H); TGN38 (I); CD63 (J); or RAB7 (K) in WT, Nbr1^−/−, and Sqstm1^−/− HSCs stimulated with cGAMP (n = 40 cells). Scale bar, 10 µm. (L) Scheme showing the delayed STING trafficking in Nbr1^−/− compared with WT HSCs. Immunoblot experiments are representative of two independent experiments. Results are presented as mean ± SEM. ***p < 0.001, ****p < 0.0001. See also Figure S4.

Figure 5.. The competitive binding of NBR1 and p62 regulates STING signaling

(A and B) Immunoblotting of cell lysate and STING (A) or p62 (B) immunoprecipitates of HSCs stimulated with cGAMP for 3 h. (C and D) Immunofluorescence staining and quantification of STING with NBR1 (C) or p62 (D) in WT HSCs stimulated with cGAMP for 2 h (n = 35 cells) Scale bar, 10 µm. (E and F) PLA and quantification of NBR1-STING (E) or p62-STING (F) in HSCs stimulated with cGAMP for 3 h (n = 21–26 cells). Scale bar, 20 µm. (G) Immunoblotting of cell lysate and STING immunoprecipitates of HEK293T cells transfected with the indicated plasmids. (H) Immunoblotting of cell lysate and STING immunoprecipitates in WT and Sqstm1^−/− HSCs stimulated cGAMP for 2.5 h. (I) Immunoblotting of cell lysate and FLAG immunoprecipitates of HEK293 cells transfected with the indicated plasmids and stimulated with cGAMP. (J) Immunofluorescence staining and quantification of NBR1 and p62 in HSCs stimulated with cGAMP (n = 35 cells). Scale bar, 10 µm. (K) Model showing the competitive binding of NBR1 and p62 to STING after cGAMP stimulation and the formation of NBR1-p62 and STING-p62 complexes. (L) (1^st-IP) Immunoblotting of cell lysate and FLAG immunoprecipitated in HEK293 cells, transfected with the indicated plasmids, and stimulated with cGAMP for 3 h. (2^nd-IP) Immunoblotting of hemagglutinin (HA) immunoprecipitated from the eluted proteins from 1^st-IP. (M) Scheme showing the interaction domain mapping of STING with p62 or NBR1. (N and O) Immunoblots in WT, Sqstm1^−/−, and Nbr1^−/− HSCs, treated with cGAMP for 4 h (N), or for 2 h (O), and analyzed by non-reducing or reducing SDS-PAGE. (P) Immunoblotting of cell lysate and STING immunoprecipitates of HEK293T cells transfected with the indicated plasmids. Immunoblot experiments are representative of two independent experiments. Results are presented as mean ± SEM ***p < 0.001, ****p < 0.0001. See also Figure S5.

Figure 6.. NBR1 and p62 regulate the STING ubiquitination by TRIM32

(A) Immunoblotting of cell lysate and STING immunoprecipitates of WT, Nbr1^−/−, and Sqstm1^−/− HSCs stimulated with cGAMP for 2.5 h. (B) Immunoblotting of cell lysate and STING immunoprecipitates of EV, NBR1 OV, and p62 OV HSCs treated with BafA1 (20 nM) for 2.5 h. (C) Volcano plot of biotinylated proteins NBR1-BioID2 versus empty-BioID2 in HSCs (n = 3 biological replicates). (D) Immunoblotting of cell lysate and TRIM32-tagged immunoprecipitates of HSCs. (E) Immunoblotting of cell lysate and STING immunoprecipitates of WT, Nbr1^−/− HSCs transduced with siRNAs. (F and G) Immunoblots in WT, Nbr1^−/− (F), or EV and p62 OV (G) HSCs, transduced with siRNAs and stimulated with cGAMP. (H and I) Immunoblotting of cell lysate and STING immunoprecipitates of WT, Nbr1^−/− (H), or Sqstm1^−/− (I) HSCs and stimulated with cGAMP for 2.5 h. (J and K) Immunoblotting of cell lysate and HA-tagged immunoprecipitates of HEK293 cells transfected with plasmids and stimulated with cGAMP for indicated times (J) or for 2.5 h (K). (L) Immunofluorescence staining and quantification of TRIM32 and NBR1 in WT HSCs stimulated with cGAMP for 2 h (n = 35 cells). Scale bar, 10 µm. (M and N) Immunoblotting of cell lysate and STING immunoprecipitates of HEK293 cells transfected with plasmids and stimulated with cGAMP for 2.5 h (M) or indicated times (N). (O) Model showing the regulation of STING-mediated signaling involving NBR1, TRIM32, and p62. Under basal conditions, NBR1 prevents the interaction between STING and TRIM32. After cGAMP stimulation, p62 binds to STING and NBR1, displacing NBR1 from its inhibitory position, allowing TRIM32 to interact with and ubiquitinate STING, initiating the STING-mediated signaling cascade. Immunoblot experiments are representative of two independent experiments. Results are presented as mean ± SEM ****p < 0.0001. See also Figure S6.

Figure 7.. NBR1 is upregulated in HSCs during HCC progression and correlates with low STING levels

(A and B) Immunofluorescence staining of NBR1, p62, and αSMA in normal liver or livers from MASH and HCC patients (A) and percentage of p62 or NBR1 positive in αSMA⁺ cells (n = 13–16 fields per sample). Scale bar: 50 µm in (B). (C–G) Analysis of HCC samples. Experimental design, baseline clinical and pathological characteristics, and the expression levels of NBR1 (C); immunofluorescence staining of NBR1, STING, and αSMA staining (scale bar, 100 µm) (D); contingency analysis of the correlation between expression of NBR1 and STING in HSCs (E and F); and multivariable logistic regression analyses for the factors associated with NBR1 expression in HSCs (n = 287) (G). Quantitative data are presented as box and whisker graphs indicating the median and 25^th and 75^th percentiles, with minimum and maximum values at the extremes of the whiskers. p values were calculated with one-way ANOVA and post hoc Tukey’s test (B), or chi-squared test (F and G). *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.