NT157 Inhibits HCC Migration via Downregulating the STAT3/Jab1 Signaling Pathway

Abstract

Purpose: The high fatality-to-case ratio of hepatocellular carcinoma is directly related to metastasis. The signal transducer and activator of transcription-3 is a key mediator of the cytokine and growth factor signaling pathways and drives the transcription of genes responsible for cancer-associated phenotypes. However, so far, no specific inhibitor for signal transducer and activator of transcription-3 has been used in clinical practice. Therefore, targeting the signal transducer and activator of transcription-3 for cancer therapy is highly desired to improve outcomes in patients with hepatocellular carcinoma.

Experimental design: Using the small-molecule inhibitor NT157, the effect of signal transducer and activator of transcription-3 inhibition on cell migration was tested in hepatocellular carcinoma cell lines and a lung metastasis model of the disease.

Results: NT157 significantly inhibited the migration of hepatocellular carcinoma cell lines in vitro and lung metastasis of hepatocellular carcinoma in vivo. Mechanistically, it inhibited the phospho-signal transducer and activator of transcription-3 in a dose- and time-dependent manner. Furthermore, NT157 treatment suppressed the c-Jun activation domain-binding protein-1 levels in the nucleus but no significant decrease was observed in its expression in the cytoplasm. Finally, high mRNA expression levels of signal transducer and activator of transcription-3 and c-Jun activation domain-binding protein-1 in hepatocellular carcinoma were associated with significantly low survival rates.

Conclusion: NT157 inhibits hepatocellular carcinoma migration and metastasis by downregulating the signal transducer and activator of transcription-3/c-Jun activation domain-binding protein-1 signaling pathway and targeting it may serve as a novel therapeutic strategy for the clinical management of hepatocellular carcinoma in the future.

Keywords: Jab1; NT157; STAT3; hepatocellular carcinoma; metastasis; migration.

Figure 1.

NT157 inhibits HCC cell line migration in vitro and their lung metastasis in vivo. A, NT157 inhibits migration of HCC cells as shown by wound healing. Serum-starved HuH-7, MHCC-97 H, SMMC-7721, and MHCC-97 L cells were treated with or without 2 μM NT157, and wound healing of these cells was detected after 24 hours. B, NT157 inhibits migration of HCC cells as shown by transwell assay. Serum-starved HuH-7, MHCC-97 H, SMMC-7721, and MHCC-97 L cells were seeded in the upper chambers of transwell system with or without 24 h 2 μM NT157 treatment. The numbers of migrated cells were counted. C, NT157 inhibits HCC metastasis in vivo. The nude mice injected with SMMC-7721 cells (1 × 106 in 100 µL PBS) were sacrificed after 4 weeks with or without NT157 treatment (100 mg/kg, intraperitoneal injection). The incidences of lung metastasis were counted.

Figure 2.

NT157 inhibits activation of STAT3/Jab1 signaling. A, NT157 inhibited p-STAT3 in HCC cells in a dose-dependent manner. Serum-starved HepG2 and SMMC-7721 cells were treated with NT157 as indicated and either stimulated with IL-6 (50 ng/mL) or not, before lysis. B, NT157 inhibited p-STAT3 in HCC cells in a time-dependent manner. Serum-starved HepG2 and SMMC-7721 cells were treated with NT157 as indicated and either stimulated with IL-6 (50 ng/mL) or not before lysis. C, NT157 inhibited p-STAT3 and Jab1 in nucleus. Serum-starved HepG2 cells were treated with NT157 (2 μM) for 8 h, and nucleus protein and plasma protein were extracted and analyzed. D, Jab1 expression was slightly decreased in plasma protein. E, The effect of NT157 against p-STAT3 was not entirely ERK-dependent. Serum-starved HepG2 cells were treated with PLX4720 for 30 min followed by NT157 (2 μM) treatment for 8 h. The cells were then lysed and analyzed by western blotting.

Figure 3.

Expression patterns of STAT3 and Jab1 in normal liver tissues and HCC tissues. The clinical data were downloaded from TCGA. A, Heat-map of STAT3 and Jab1 expression. B, STAT3 and Jab1 expression in normal liver tissues and HCC tissues. C, Correlation of STAT3 and Jab1 expression. D, Kaplan-Meier analyses of the association between STAT3 or Jab1 protein expression and survival. The patients were stratified into high expression group and low expression group according to the optimal cutoff value of mRNA. E, Kaplan-Meier analyses of the association between combined STAT3 and Jab1 protein expression and survival.