Cancer Res Treat.  2019 Jul;51(3):886-900. 10.4143/crt.2018.375.

Jab1 Silencing Inhibits Proliferation and Sensitizes to Cisplatin in Biliary Tract Cancer

• Nam AR
• Kim JW
• Park JE
• Bang JH
• Jin MH
• Oh DY
• Bang YJ

• KMID: 2454281
• DOI: http://doi.org/10.4143/crt.2018.375

Abstract

PURPOSE
Jab1 is a coactivator of c-Jun that enhances the transcriptional function of c-Jun. Jab1 is frequently overexpressed in various cancers and is associatedwith poor prognosis of cancer patients. Thus, Jab1 could be a potential therapeutic target in cancer. However, the role of Jab1 in biliary tract cancer (BTC) has not been studied.
MATERIALS AND METHODS
We performed in vitro and in vivo experiments to evaluate the therapeutic potential ofJab1 inhibition in BTC.
RESULTS
Among 8 BTC cell lines, many showed higher Jab1 expression levels. In addition, Jab1 silencing by siRNA increased p27 expression levels. SNU478 and HuCCT-1 cells exhibited profound Jab1 knockdown and increased p27 expression by Jab1-specific siRNA transfection. Jab1 silencing induced anti-proliferative and anti-migratory effects and resulted in G1 cell cycle arrest in SNU478 and HuCCT-1 cells. In addition, Jab1 silencing potentiated the anti-proliferative and anti-migratory effects of cisplatin by increasing DNA damage. Interestingly,Jab1 knockdown increased PTEN protein half-life, resulting in increased PTEN expression. In the HuCCT-1 mouse xenograft model, stable knockdown of Jab1 by shRNA also showed anti-proliferative effects in vivo, with decreased Ki-67 expression and AKT phosphorylation and increased Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and p27 expression.
CONCLUSION
Jab1 knockdown demonstrated anti-proliferative and anti-migratory effects in BTC cells by increasing DNA damage and stabilizing PTEN, resulting in G1 cell cycle arrest. In addition, Jab1 silencing potentiated the anti-proliferative effects of cisplatin. Our data suggest that Jab1 may be a potential therapeutic target in BTC that is worthy of further investigations.

Keyword

Jab1; Biliary tract neoplasms; Proliferation; Therapeutic target; Cisplatin

MeSH Terms

Animals
Biliary Tract Neoplasms*
Biliary Tract*
Cell Line
Cisplatin*
DNA Damage
G1 Phase Cell Cycle Checkpoints
Half-Life
Heterografts
Humans
In Vitro Techniques
Mice
Phosphorylation
Prognosis
PTEN Phosphohydrolase
RNA, Small Interfering
Transfection
Cisplatin
PTEN Phosphohydrolase
RNA, Small Interfering

Figure

• 

  Fig. 1. Effects of Jab1 on p27 expression and cell proliferation in biliary tract cancer (BTC) cell lines. (A) Basal expression levels of Jab1 and p27 in eight BTC cell lines were analyzed by western blot and quantified by ImageJ software. (B) Effects of Jab1 silencing by siRNA on p27 expression were analyzed and quantified in eight BTC cell lines. (C) Knock-down effects of Jab1-specific siRNA were evaluated at 1 day, 3 days, and 5 days in SNU478 and HuCCT-1 cells. (D) MTT assays (upper) and colony forming assays (lower) were performed to evaluate the effects of Jab1 silencing on proliferation of SNU478 and HuCCT-1 cells. **p < 0.01.

• 

  Fig. 2. Effects of Jab1 silencing on cell cycle and migration. (A) Cell cycle analysis was performed in SNU478 and HuCCT1 cell lines after transfection with Jab1-specific or control siRNAs. **p < 0.01. (B) Cyclin D1/E/A expression levels were analyzed by western blot in SNU478 and HuCCT-1 cell lines transfected with Jab1-specific or control siRNA (C) Wound healing assays were performed to evaluate the effects of Jab1 silencing on migration in SNU478 and HuCCT-1 cell lines. *p < 0.05. (D) Transwell migration assays were performed to investigate the effects of Jab1 silencing on invasion of SNU478 and HuCCT-1 cells. **p < 0.01.

• 

  Fig. 3. Jab1 silencing and cisplatin sensitivity. (A) The effects of Jab1 silencing on the anti-proliferative activity of cisplatin at various concentrations were evaluated by colony formation assays in SNU478 and HuCCT-1 cells. Jab1-specific or control siRNA-transfected cells were treated with cisplatin at a concentration of 0, 0.5, 1.0, and 2.0 μM for 10 days. **p < 0.01. (B) Wound healing assays demonstrated the effects of Jab1 silencing on anti-migratory activity of cisplatin at a concentration of 0, 1.0, 2.5, and 5.0 μM. **p < 0.01. ***p < 0.001. (C) Comet assays were performed to quantify the effects of Jab1 silencing on DNA damage in SNU478 and HuCCT-1 cells. **p < 0.01.

• 

  Fig. 4. Effects of Jab1 on PTEN protein half-life. (A) The effects of Jab1 silencing on PTEN protein and mRNA expression levels were evaluated by western blot and real-time quantitative PCR in SNU478 and HuCCT-1 cells. N.S., not significant. The effects of Jab1 on Src and AKT and their phosphorylation (Src Tyr416 and AKT Ser473) were also analyzed by western blot. (B) In SNU478 and HuCCT-1 cell lines after transfection with Jab1-specific or control siRNA, PTEN protein expression levels were analyzed by western blot (upper) at 0, 1, 2, 6, and 24 hours of cycloheximide treatment. The band intensity (lower) was quantified using ImageJ software and normalized by β-actin expression levels.

• 

  Fig. 5. Stable knockdown of Jab1 by shRNA in HuCCT-1 cells. (A) Western blot analysis revealed the effects of stable knockdown of Jab1 by shRNA on p27 expression levels in HuCCT-1 cells. (B) MTT assays were performed to evaluate the effects of Jab1 knockdown on proliferation. (C) Wound healing assays demonstrated the anti-migratory effects of stable knockdown of Jab1 by shRNA in HuCCT-1 cells. **p < 0.01. (D) Using the HuCCT-1 mouse xenograft model, the effects of stable Jab1 silencing on proliferation were evaluated in vivo. **p < 0.01. (E) The tumors were harvested and analyzed by immunohistochemical staining of Jab1, Ki-67, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. (F) Western blot analysis evaluated the effects of Jab1 knockdown by shRNA on Jab1 and p27 expression, AKT and ERK phosphorylation, and cyclin D1 and A expression in vivo. The band intensity (lower) was quantified using ImageJ software and normalized by β-actin expression levels.

Reference

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