Dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes hepatobiliary carcinogenesis in non-alcoholic fatty liver disease

Abstract

Background & aims: Non-alcoholic fatty liver disease (NAFLD), the hepatic correlate of the metabolic syndrome, is a major risk factor for hepatobiliary cancer (HBC). Although chronic inflammation is thought to be the root cause of all these diseases, the mechanism whereby it promotes HBC in NAFLD remains poorly understood. Herein, we aim to evaluate the hypothesis that inflammation-related dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes HB carcinogenesis.

Methods: We use murine NAFLD models, liver biopsies from patients with NAFLD, human liver cancer registry data, and studies in liver cancer cell lines.

Results: Our results confirm the hypothesis that inflammation-related dysregulation of the ESRP2-NF2-YAP/TAZ axis promotes HB carcinogenesis, supporting a model whereby chronic inflammation suppresses hepatocyte expression of ESRP2, an RNA splicing factor that directly targets and activates NF2, a tumor suppressor that is necessary to constrain YAP/TAZ activation. The resultant loss of NF2 function permits sustained YAP/TAZ activity that drives hepatocyte proliferation and de-differentiation.

Conclusion: Herein, we report on a novel mechanism by which chronic inflammation leads to sustained activation of YAP/TAZ activity; this imposes a selection pressure that favors liver cells with mutations enabling survival during chronic oncogenic stress.

Lay summary: Non-alcoholic fatty liver disease (NAFLD) increases the risk of hepatobiliary carcinogenesis. However, the underlying mechanism remains unknown. Our study demonstrates that chronic inflammation suppresses hepatocyte expression of ESRP2, an adult RNA splicing factor that activates NF2. Thus, inactive (fetal) NF2 loses the ability to activate Hippo kinases, leading to the increased activity of downstream YAP/TAZ and promoting hepatobiliary carcinogenesis in chronically injured livers.

Keywords: alternative RNA splicing; epithelial splicing regulatory protein-2 (ESRP2); hepatocellular carcinoma (HCC); hippo kinase; liver cancer; neurofibromatosis-2 (NF2); nonalcoholic fatty liver disease (NAFLD); yes-associated protein (YAP).

Fig. 1.. ESRP2 expression is inversely related to severity of liver inflammation, liver injury and systemic glucose intolerance in mice with NASH.

Mice were fed chow or choline-deficient, L-amino acid defined, high-fat diet (CDAHFD) for 22 weeks to induce nonalcoholic steatohepatitis (NASH). (A) qPCR for Tnfα in liver; (B) serum alanine transaminase (ALT), aspartate transaminase (AST); (C) hemoglobin A1c (HbA1c); (D) qPCR for Esrp2 in liver; (E) Immunoblots for ESRP2 in liver lysates normalized to GAPDH; (F) Liver immunohistochemistry (IHC) for ESRP2 (Scale bars = 100 μm). Results are graphed as dot plots (Chow: white circle, CDAHFD: blue square) with mean±s.e.m. (red bars); student t test was used for statistics (n=5 mice/group). *p < 0.05, **p < 0.005.

Fig. 2.. Fetal Nf2 variants accumulate, activating YAP/TAZ and promoting adult-to-fetal reprogramming in mice with NASH.

(A) Alternative splicing of Nf2 in livers of chow- and CDAHFD-fed mice. The larger (upper) band includes exon 16 (45 nucleotides in length), while the smaller (lower) band lacks this exon. Differences in percent spliced in (PSI) values are shown. (B) Immunoblots for MST1/2, phosphorylated YAP (p-YAP), total YAP and TAZ normalized to GAPDH in liver lysates. (C) qPCR for YAP/TAZ targets (Areg and Birc5), a microRNA suppressed by YAP (Let-7g-5p), an oncofetal gene that is regulated by Let-7 (Igf2bp3), stem/progenitor cell markers (Krt7, Sox9, Gli2), and a mature hepatocyte marker (C/ebpα); (D) Immunoblots for KRT7, GLI2 and C/EBPα normalized to GAPDH. Results are shown as dot plots (Chow: white circle, CDAHFD: blue square) with mean±s.e.m. (red bars); student t test was used for statistics (n=5 mice/group). *p < 0.05, **p < 0.005. (E) Liver IHC for KRT7 (Scale bars = 100 μm).

Fig. 3.. ESRP2 suppression, fetal NF2 variant enrichment, and YAP/TAZ activation in human NASH and liver cancer.

(A) Ordinal regression analysis of published data shows that ESRP2 gene expression profiles vary with NAFLD activity score (NAS) in humans. (B) Alternative splicing of NF2 in livers from individuals with normal livers (n=12), NAFLD (n=47), or HCC (n=10). PSI values are shown as mean±s.e.m; student t test was used. *p < 0.05, **p < 0.005. (C) IHC for ESRP2 in liver from patient with NASH-cirrhosis and NASH-driven CCa. Scale bars = 100 μm. (D) Alternative splicing of NF2 in human NASH cirrhosis (non-tumor) and NASH-CCa (tumor). PSI values are beneath blots. (E) UMAP plot of NASH-CCa isolate. (F) Distribution of YAP/TAZ targets (BIRC5, CTGF, CYR61) and ESRP2 among the 13 NASH-CCa clusters analyzed by scRNA-seq. (G) Representative images of cancer cells isolated from NASH-CCa and cultured on standard tissue culture plates (2D) or ultra-low attachment plates for anchorage-independent spheroid formation (3D). Tumor spheroids were sorted by size (smaller or larger than 40 μm diameter); scale bars = 100 μm. (H) Representative H&E and immunofluorescent staining of tumor spheroids for YAP, CD133, and DAPI; scale bars = 50 μm. (I) Bromodeoxyuridine (BrdU) incorporation in NASH-CCa tumor spheroids before and after treatment with verteporfin (VP), an inhibitor of YAP/TAZ. Either fetal bovine serum (FBS) or vehicle (Veh, 0.1% DMSO) were used as controls. Representative images of tumor spheroids before (Veh) and after VP (2 μM) treatment are shown; scale bars = 100 μm. UMAP, uniform manifold approximation and projection; DAPI, 4’,6-diamidino-2-phenylindole.

Fig. 4.. Fetal NF2 variant enrichment promotes neoplastic growth of liver cancer cells.

Huh-7 cells were transduced with GFP-tagged (AdGFP) or ESRP2-overexpressing (AdESRP2) adenoviruses for 24 or 48 hours (n=3 repeats/group/time). (A) qPCR for ESRP2; (B) Immunoblots for ESRP2 normalized to GAPDH in cells harvested after 48 hours; (C) Alternative splicing of NF2; (D) Immunoblots for YAP and TAZ normalized to GAPDH; (E) qPCR for YAP/TAZ targets (AREG, CTGF, CYR61, PTGS2). (F) Representative images of AdGFP and AdESRP2 transduced Huh-7 cell tumorspheres on culture day 6. Scale bars = 100μm. Size of tumor spheroids was assessed using Image J 1.8.0 software. Results are graphed as dot plots (AdGFP: white circle, AdESRP2: blue square) with mean±s.e.m. (red bars), and statistical analyses were performed by one-way ANOVA with Tukey’s corrections or using student t test. *p < 0.05, **p < 0.005.

Fig. 5.. Fetal NF2 variant enrichment promotes neoplastic growth of liver cancer cells.

(A-D) CRISPR-Cas9 was used to remove exon 16 from NF2 in Huh-7 cells (ΔE16). Results from three experimental replicates are shown. (A) Alternative splicing of NF2 in parental Huh-7 cells and two different ΔE16 clones; (B) qPCR for YAP/TAZ targets (AREG, CTGF, CYR61, PTGS2), a stem cell marker (SOX4) and a mature hepatocyte marker (C/EBPα) in parental WT Huh-7 cells and the second clone of Huh-7 ΔE16 cells. (C) Immunoblots for p-LATS1 on either Ser909 or Thr1079 residue, total LATS1, p-MST1 on Thr183/ p-MST2 on Thr180, total MST1, p-YAP on Ser127, p-TAZ on Ser89 and total YAP/TAZ normalized to GAPDH in WT and ΔE16 Huh-7 cells cultured in anchorage-independent conditions for 11 days. (D) Representative images of WT and ΔE16 Huh-7 cell tumorspheres on culture day 11. Scale bars = 100μm. Tumor spheroids (>75 μm) were counted on culture day 6 (n=3/group); size was assessed on culture day 11 using Image J 1.8.0 software. Results are graphed as dot plots (Parental Huh-7: white circle, Huh-7 ΔE16: blue square) with mean±s.e.m. (red bars), and statistical analyses were performed by one-way ANOVA with Tukey’s corrections or using student t test. *p < 0.05, **p < 0.005.

Fig. 6.. Fetal NF2 variant enrichment and ESRP2 reduction correlate with poor survival in HCC patients.

(A) Representative image of IHC for ESRP2 in liver sections from patient with NASH-associated HCC. Scale bars = 100 μm. (B) Analysis of ESRP2 expression in HCC samples (n=369) of The Cancer Genome Atlas (TCGA) dataset compared to non-tumor samples (n=160) from TCGA and GTEx datasets. Expression is shown as log₂(TPM+1) with median; values > |log₂FC| = 1 and p-value <0.05 are considered to be differentially expressed. (C) ESRP2 gene expression in HCC tumor samples from the TGCA data set grouped by recurrence (No: n=51, Yes: n=20). Data is shown as log₂(x+1) RPKM with median, student t test was used for statistics. *p < 0.05. (D) Kaplan-Meier plots demonstrate the effect of ESRP2 gene expression on survival. Plots were generated using KM-plotter (https://kmplot.com). (E) PSI values of NF2 exon 16 in HCCs (n=417) from TCGA SpliceSeq dataset compared to the respective adjacent non-tumor livers. (F) Expression of an NF2 variant excluding exon 16 (ENST00000338641.8) in HCC (n=369) tumor samples from TCGA dataset compared to non-tumor samples (n=160) from TCGA and GTEx datasets. Data are shown as log₂(TPM+1) with median; genes with > |log₂FC| = 1 and p-value <0.05 are considered to be differentially expressed. (G) Correlation between the level of adult NF2 splicing variant and ESRP2 level in TCGA HCC samples. Point plot illustrating the correlation between NF2 exon 16 splicing (PSI) and ESRP2 expression. Pearson correlation (r) and p-value (p) are shown. The X-axis shows the log2 aggregate expression level of ESRP2 as quantified by normalized read counts. The Y-axis shows the NF2 exon 16 PSI (linear). (H) Overall and disease-free survival analyses of HCC patients in the TCGA dataset based on mRNA expression level of the fetal variant of NF2 (ENST00000338641.8). TPM, transcripts per million; RPKM, reads per kilobase per million mapped reads; HR, hazard ratio.