Therapeutic blockade of ER stress and inflammation prevents NASH and progression to HCC

Abstract

The incidence of hepatocellular carcinoma (HCC) is rapidly rising largely because of increased obesity leading to nonalcoholic steatohepatitis (NASH), a known HCC risk factor. There are no approved treatments to treat NASH. Here, we first used single-nucleus RNA sequencing to characterize a mouse model that mimics human NASH-driven HCC, the MUP-uPA mouse fed a high-fat diet. Activation of endoplasmic reticulum (ER) stress and inflammation was observed in a subset of hepatocytes that was enriched in mice that progress to HCC. We next treated MUP-uPA mice with the ER stress inhibitor BGP-15 and soluble gp130Fc, a drug that blocks inflammation by preventing interleukin-6 trans-signaling. Both drugs have progressed to phase 2/3 human clinical trials for other indications. We show that this combined therapy reversed NASH and reduced NASH-driven HCC. Our data suggest that these drugs could provide a potential therapy for NASH progression to HCC.

Fig. 1.. daHeps are enriched in NASH before HCC and characterized by the exacerbated ER stress–related gene expression program.

(A) Uniform manifold approximation and projection (UMAP) visualization of reclustered hepatocytes. Four subsets identified representing hepatocyte zonation phenotypes (Zone_1_Hep, Zone_2_Hep, and Zone_3_Hep) and daHep. (B) Expression distribution of (left) zonation marker genes and (right) up-regulated and down-regulated daHep markers in the UMAP space. (C) Mapping of daHep scores generated from Carlessi et al. (15) onto the reclustered hepatocytes UMAP. (D) (Top left) Hepatocytes UMAP visualization split by HCC outcome at 40 weeks (TB, tumor-bearing; TF, tumor-free). (Top right) Relative frequencies of hepatocyte subsets in each sample. (Bottom) Box and whisker plots showing frequencies of each hepatocyte subset according to HCC outcome. *P < 0.05, ***P < 0.01 by unpaired t test, TF, n = 4 and TB, n = 6. (E) Heatmap showing average expression of 243 mouse orthologs of human UPR program from Reich et al. (40) across hepatocyte subsets. (F) Mapping of UPR scores generated from Reich et al. (40) across hepatocyte subsets: (left) violin plot and (right) UMAP visualization. (G) Expression distribution of ER stress markers in the UMAP space, showing high levels where daHep cells localize. (H) (Top) GSEA of log₂FC ranked daHep DEGs showing the top 10 over- and underrepresented gene ontology (biological process) terms. (Bottom) Enrichment plots for “response to ER stress” and “lipid catabolic process” gene sets.

Fig. 2.. Treatment of MUP-uPA/sgp130Fc mice with BGP-15 ameliorates markers of NASH.

Liver biopsy samples were obtained at 24 weeks. Representative H&E staining in WT, MUP-uPA that developed tumors and MUP-uPA/sgp130Fc BGP-15–treated mice that did not develop tumors (A). Quantification of average lipid droplet area (B), steatosis (C), hepatocyte ballooning (D), lobular inflammation (E), and NAS (F). The NAS is a measure of grade and is the sum of numerical scores applied to steatosis, hepatocellular ballooning, and lobular inflammation. Collagen deposition in WT, MUP-uPA, and MUP-uPA/sgp130Fc BGP-15–treated mice measured by PSR. Representative images (G), average collagen size (H), and fibrosis score (I). Fibrillar collagen measured by SHG microscopy (J). Representative images (top), intensity SHG signal versus depth of image (bottom left), and SHG signal at peak (bottom right). Geometrical arrangement of collagen fibrillar bundles measured by GLCM. (K) Representative images (top), correlation versus distance (bottom left), and mean correlation (bottom right). For (B), (C), (E), (F), (H), and (I): One-way analysis of variance (ANOVA) was performed. (J and K) Unpaired Welch’s t tests compared with MUP-uPA. The following numbers of biological replicates were used (independent mice) per group in each experiment: (B) 4 to 7; (C to F) 10 to 15; (H) 3 to 8; (I) 13 to 14; (J and K) 13 to 17. Data are expressed as means ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 3.. Treatment of MUP-uPA/sgp130Fc mice with BGP-15 ameliorates NASHdriven HCC.

- Liver samples were obtained at 40 weeks. Tumor incidence in WT mice, MUP-uPA mice, and MUP-uPA/sgp130Fc mice treated with BGP-15 (A). Representative liver images (B). Tumor number (C), maximal tumor volume (D), and representative images (E). Representative H&E staining in WT, MUP-uPA, and MUP-uPA/sgp130 BGP-15–treated mice (F). Quantification of average lipid steatosis, lobular inflammation, hepatocyte ballooning, and NAS (G). The NAS is a measure of grade and is the sum of numerical scores applied to steatosis, hepatocellular ballooning, and lobular inflammation. Representative images (H) and quantification (I) for TUNEL staining. (C) and (D) were analyzed by unpaired t tests. (G) to (I) were analyzed by one-way ANOVA. The following numbers of biological replicates were used (independent mice) per group in each experiment: (A) 18 to 22; (C and D) 3 to 10; (F and G) 4 to 10. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.. Treatment of MUP-uPA/sgp130Fc mice with BGP-15 reduces inflammation and ER stress.

Liver samples were obtained at 40 weeks. RT-PCR analyses of Calr, a gene that encodes calreticulin; Pdia6, a gene that inhibits aggregation of misfolded proteins; hsp90aa1, a gene that encodes HSP90; dnajc3, a gene that encodes a protein that inhibits PKR; ppp1r3g, a gene that encodes protein phosphatase 1 regulatory subunit 3G; and ddit4l, a gene encoding DNA damage-inducible transcript 4–like protein, which inhibits cell growth by regulating the TOR signaling pathway (A). Representative image and quantification of XBP-1 mRNA splicing (B). Representative Western blot and quantification of CHOP (C), HSF1, p-eIF2α (D), p-JNK (E), and calreticulin (F). RT-PCR analyses of TNF (G), IL-4 (H), and EMR1, the gene that encodes the F4/80 protein (I). Unpaired t tests were used to analyze all data. The following numbers of biological replicates were used (independent mice) per group in each experiment: (A) 4; (B to F) 6 to 7; (G and I) 6; (H) 7 to 12. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.. MUP-uPA/sgp130Fc mice treated with BGP-15 do not show reduced liver lipids.

Liver and blood samples were obtained at 40 weeks. Total lipids (A), glycerolipids (B), phospholipids (C), lysophospholipids (D), plamalogens (E), sphingolipids (F), ceramides (G), sterols (H), and free cholesterol (I) in liver samples and triglycerides (J), free (K) and total (L) cholesterol in high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in plasma samples in WT mice, MUP-uPA mice, and MUP-uPA/sgp130 mice treated with BGP-15. One-way ANOVA with Tukey’s multiple comparisons where indicated. The following numbers of biological replicates were used (independent mice) per group in each experiment: (A to I) 10 to 18; (J to L) 6 to 10. Data are expressed as means ± SEM. **P < 0.01.

Fig. 6.. Treatment with BGP-15 and sgp130Fc after NASH onset results in disease regression.

WT and MUP-uPA mice were untreated or treated with BGP-15 [in drinking water and sgp130Fc (twice weekly injections of 0.5 mg/kg)] for 11 weeks (DT). Representative images of H&E (A) and Picosirus Red (PSR) (C) and quantification of fibrosis (B) and lobular inflammation (D) in WT and MUP-uPA mice at 12 weeks before DT or placebo treatment. Representative images of H&E (E) and quantification steatosis (F), ballooning hepatocytes (G), lobular inflammation (H), and NAS (I) in WT and MUP-uPA mice either untreated or DT. The NAS is a measure of grade and is the sum of numerical scores applied to steatosis, hepatocellular ballooning, and lobular inflammation. Data in (F) to (I) represent the change in score from biopsy (pre-treatment) to cull (post-treatment). Representative PSR images (J) and quantification of % fibrosis (K) and change in fibrosis area (pre-post treatment) (L) in WT, MUP-uPA, and MUP-uPA/sgp130 BGP-15–treated mice. (B) and (D), unpaired t tests; (F) to (I), (K), and (L), one-way ANOVA with Tukey’s multiple comparisons where indicated. The following numbers of biological replicates were used (independent mice) per group in each experiment: (B and D) 26 to 38; (F to I) 7 to 24; (K and L) 8 to 22. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. In (L), P = 0.07 relative to WT DT.