TM4SF5-mediated liver malignancy involves NK cell exhaustion-like phenotypes

Abstract

Aberrant extracellular matrix and immune cell alterations within the tumor microenvironment promote the pathological progression of liver carcinogenesis. Although transmembrane 4 L six family member 5 (TM4SF5) is involved in liver fibrosis and cancer, its mechanism avoiding immune surveillance during carcinogenesis remains unknown. We investigated how TM4SF5-mediated signaling caused immune evasion using in vitro primary cells and in vivo liver tissues from genetic or chemically induced mouse models. TM4SF5-transgenic and diethylnitrosamine (DEN)-induced liver cancer mouse models exhibited fibrotic and cancerous livers, respectively, with enhanced TM4SF5, pY⁷⁰⁵STAT3, collagen I, and laminin γ2 levels. These TM4SF5-mediated effects were abolished by TM4SF5 inhibitor, 4'-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC). TM4SF5-dependent tumorigenesis involved natural killer (NK) cell exhaustion-like phenotypes including the reduction of NK cell number or function, which were blocked with TSAHC treatment. TM4SF5 expression in cancer cells downregulated stimulatory ligands and receptors for NK cell cytotoxicity, including SLAMF6, SLAMF7, MICA/B, and others. TM4SF5 suppression or inhibition reduced STAT3 signaling activity and recovered the receptor levels and NK cell surveillance, leading to reduced fibrotic and cancerous phenotypes, and longer survival. Altogether, these findings suggest that TM4SF5-mediated STAT3 activity for extracellular matrix modulation is involved in the progression of liver disease to HCC and that TM4SF5 appears to suppress NK cells during liver carcinogenesis.

Keywords: Immune checkpoint; L6 family member; Liver cancer; NK cell immune therapy; Signal transduction.

Fig. 1

TM4SF5-mediated hepatic carcinogenesis in TM4SF5-transgenic mice. A Liver nodules in 1-year-old FVB/N-Tg^TM4SF5 mice (n = 2/7) compared with age-matched wild-type (WT) mice (n = 0/7). Tg^TM4SF5 mice showed enlarged spleens compared with age-matched WT mice. B The livers of 1-year-old FVB/N-Tg^TM4SF5 mice were examined for aspartate aminotransferase (AST), alanine aminotransferase (ALT), low-density lipoprotein (LDL), albumin, and serum triglyceride (TG) levels and were compared with those of age-matched WT mice. C, D The mRNA levels of the livers of 1-year-old FVB/N-Tg^TM4SF5 and age-matched WT mice were analyzed for the indicated molecules. For statistical significance, * and *** represent p ≤ 0.05 and p ≤ 0.001, respectively, and ns indicates no significance, following Student’s t tests. E, F The livers of 1-year-old FVB/N-Tg^TM4SF5 and age-matched WT mice were processed for immunoblot (E) and immunohistochemical (F) analyses. The livers of Tg^TM4SF5 mice showed enhanced expression of hepatocellular carcinoma markers, including CD34, α-fetoprotein (AFP), and α-l-fucosidase [FUCA (AFU)], in addition to pY⁷⁰⁵STAT3 and extracellular matrix factors, including collagen I, laminin γ2, and laminins. Data shown represent three isolated experiments

Fig. 2

Clinical relevance of TM4SF5-mediated STAT3 activity, extracellular matrix production, and hepatic cancer. A Human liver cancer tissues were processed for immunohistochemical analysis and showed higher levels of TM4SF5, pY⁷⁰⁵STAT3, collagen I, and laminins, compared with normal surrounding liver tissues. B, C Gene expression profiles of liver cirrhosis [normal (Nor) vs. cirrhosis (Cir) vs. HCC] from GSE6764 were analyzed and showed TM4SF5-positive cirrhosis (p = 0.055) and HCC (p = 0.011) and SOCS1-reduced cirrhosis and HCC (p = 0.013) groups. p-values were calculated by the Dunnett’s test and ordinary one-way ANOVA. Other analyses of hepatocellular carcinoma (HCC) (normal vs. HCC) from GSE76427 showed TM4SF5-positive (p = 0.010) and LAMC2-promoted HCC (p = 0.016) groups compared with normal groups. p-values were calculated by the Mann–Whitney U test. D Primary hepatocytes were prepared from mice treated with CCl₄ for 16 weeks as an animal cirrhosis model and were transfected with STAT3-specific or scrambled control siRNA for 48 h before harvesting whole-cell extracts for immunoblot analysis. Data shown represent three independent experiments

Fig. 3

TM4SF5 expression and activity-dependent granulomatous inflammation during diethylnitrosamine-induced hepatic carcinogenesis. A Age-matched WT or Tm4sf5^−/− male C57BL/6 mice (2 weeks old) were intraperitoneally injected once with diethylnitrosamine (DEN, 25 mg/kg body weight) and twice per week with 4′-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC, 5.0 mg/kg body weight) for 45 weeks. After 45 weeks on a normal diet ad libitum, the mice were sacrificed for analysis. Age-matched WT and knockout mice (n = 6), DEN-treated WT and knockout mice (n = 7), and DEN/TSAHC-treated WT mice (n = 5) were examined in parallel. B Five representative liver images were shown for each group. C Tumors with a diameter larger than 1.0 mm were counted, and the largest tumor in each animal was determined. Age-matched WT and knockout animals without DEN treatment did not form any tumors. One outlier (second, left) from the DEN/TSAHC-treated WT group was not included in the graphs. p-values were calculated by Sidak’s test and one-way ANOVA. A p-value ≤ 0.05 was considered statistically significant. D After 45 weeks, spleens were also collected from each animal and weighed. Data are represented as the mean ± standard deviation. p-values were calculated by Sidak’s test and one-way ANOVA. A p-value ≤ 0.05 was considered statistically significant. E, F Liver tissues were processed for hematoxylin and eosin staining or immunohistochemical analysis for Ki67 and AFP protein expression levels. N and T depict normal and tumor regions, respectively (E). More significant granuloma-like lesions were observed in the tumor tissues of DEN-treated WT mice compared with DEN/TSAHC-treated WT mice (F). Data shown represent three independent experiments

Fig. 4

Natural killer cell involvement in diethylnitrosamine-induced, TM4SF5-dependent hepatic carcinogenesis. Spleen and liver tissues from WT and Tm4sf5^−/− animals with or without diethylnitrosamine (DEN) treatment and in the absence or presence of 4′-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC) co-treatment (described in Fig. 3A) were processed for splenic and hepatic immune cell population analysis by flow cytometry. A, B One outlier was excluded from the DEN/TSAHC-treated WT group. Splenic tissues for each experimental group were processed for immune cell population analysis using CD3⁺/CD4⁺ [T-helper (Th) cells], CD3⁺/CD8⁺ (T cells), and CD³⁻/NK1.1⁺ [natural killer (NK) cells] cell markers (A). The cell populations were graphed, and p values were calculated by Sidak’s test and one-way ANOVA. A p-value ≤ 0.05 was considered statistically significant (B). C, D One outlier was excluded from the DEN/TSAHC-treated WT group. Hepatic tissues for each experimental group were processed for immune cell population analysis using CD3⁺/CD4⁺, CD3⁺/CD8⁺, and CD3⁻/CD45⁺/NK1.1⁺ cell markers (C). The intrahepatic cell populations were graphed, and p values were calculated by Dunnett’s test and one-way ANOVA. A p-value ≤ 0.05 was considered statistically significant (D). E NK cells were analyzed for cytokine or granule secretion via antibody staining and flow cytometry. p values were calculated by Sidak’s test and one-way ANOVA. A p-value ≤ 0.05 was considered statistically significant. Data represent three independent experiments or measurements

Fig. 5

Immune evasion from natural killer cell cytotoxicity via TM4SF5 expression in hepatocytes. A–C Huh7 (A, B), Hep3B, and HepG2 cells with endogenous TM4SF5 expression (C) were infected with lentivirus for non-specific sequence (NS) or shTM4SF5 (targeting sequences #2 and #4 of TM4SF5) before expression level analysis by qRT-PCR for TM4SF5 or ligands for natural killer cell (NK) cell stimulation (A, C) or before a NK cell cytotoxicity assay using a co-culture system [at different ratios (E:T) of target (T) Huh7 hepatocytes and effector (E) NK92 cells] for 4 h (B). D–G Huh7 (D, E) or HepG2 (F, G) target (T) cells with or without TM4SF5 suppression (A, F, left) were co-cultured with NK92 effector (E) cells for 4 h at an E:T ratio of 10:1 (D, E) or the indicated ratios (F, G) for NK92 cytotoxicity analysis (F, right), qRT-PCR (D–F, left), or whole-cell extract preparation prior to immunoblot analysis (G). H For NK92 cytotoxicity analysis, Huh7 target cells and NK92 effector cells were co-cultured for 6 h (E:T = 10:1), and 4′-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC) was administered at the indicated concentrations. Statistical significance is indicated by *, **, ***, or ****, which represent a p-value ≤ 0.05, 0.01, 0.001, or 0.0001, respectively, and ns indicates no significance, but red-highlighted ns indicates 0.05 ≤ p-values ≤ 0.10. p-values were calculated by one-way analysis of variance (ANOVA) or one-tailed and unpaired Mann–Whitney U tests. I, J Whole-cell extracts from hepatocytes with different TM4SF5 expression levels (I) and from hepatocytes transiently transfected with empty vector (EV) or TM4SF5 plasmids with or without TSAHC treatment at the indicated concentrations for 24 h (J) were prepared for immunoblot analysis of the indicated molecules. MICA/B blot from stable control SNU449C_p cells showed multiple bands, although stably TM4SF5-expressing SNU449T_p or T₇ cells and other transiently transfected SNU449 cell lines showed a single band. Data shown represent three independent experiments

Fig. 6

Abolishment of diethylnitrosamine-mediated precancerous and cancerous phenotypes by TM4SF5 inhibition. A–D Five-week-old BALB/c mice (n ≥ 5) were treated with diethylnitrosamine (DEN) for 27 weeks with or without intraperitoneal (IP) injection of DMSO as the vehicle control or 4′-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC), as explained in the Materials and Methods section. TSAHC was administered either at the beginning of DEN treatment (DEN + TSAHC1) or 10 weeks after DEN treatment (DEN + TSAHC2). Liver tissues were imaged and processed for hematoxylin and eosin staining (A). Survival rates of the mice in each experimental group were graphed (B). Whole-tissue extracts were also prepared and processed for immunoblot analysis (C), or tissues were processed for immunohistochemical analysis of the indicated molecules (D). Immunoglobulins (IgG) from rabbit or mouse were used for the negative control stains without the primary antibodies. p-values were calculated by Gehan–Breslow–Wilcoxon test for *p = 0.0435. p-value > 0.05 was considered statistically insignificant (ns; p = 0.3679). Data shown represent three independent experiments. E Gene expression profiles of liver cirrhosis [normal (Nor, n = 220) vs. HCC (n = 225)] from GSE14520 were analyzed and showed HCC with significantly increased TM4SF5 (p < 0.0001), and among the samples showing the increased TM4SF5 expressing in HCC (n = 143) SLAMF7 (p < 0.0001), MICA (p = 0.0161), and ULBP1 (p < 0.0001) were significantly decreased in HCC, compared with normal counterparts. Meanwhile, ULBP2 was insignificantly decreased (p = 0.0926). p-values were calculated by the unpaired, two-tailed Student’s t tests

Fig. 7

Working models for TM4SF5-mediated suppression of NK cell surveillance. (Left) Highly expressed TM4SF5 in liver cancer cells triggers intracellular signaling for the downregulation of surface ligand-related factors at the transcriptional level, which leads to inhibited NK cell cytotoxicity [i.e., immune evasion from natural killer (NK) cells], in addition to enhanced TM4SF5-pY⁷⁰⁵STAT3 signaling and extracellular matrix production for the promotion of precancerous (fibrotic/cirrhotic) and cancerous phenotypes. (Right) Inhibition of TM4SF5 using anti-TM4SF5 compound, 4′-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC), upregulates the immune checkpoints between NK cells and hepatocytes, including SLAM, NKG2D, and MICA/B member receptors and ligands, which leads to the stimulation of NK cell cytotoxic receptors and enhanced NK cell cytotoxicity. Activation of NK cell cytotoxicity upon TM4SF5 inhibition by TSAHC treatment appeared to cause (1) inactive STAT3-mediated activation of ligands in hepatocytes stimulatory for NK cell cytotoxicity [54] and (2) ERK activation-mediated increases in stimulatory receptors in NK cells neighbored with TM4SF5-inhibited hepatocytes (Fig. 5G). TM4SF5-mediated STAT3 activation and extracellular matrix production are blocked by TSAHC treatment, which leads to NK cell cytotoxic activity as a potential immunotherapy for TM4SF5-positive liver cancer