2. Academic Validation
  3. The oncogenic role of NF1 in gallbladder cancer through regulation of YAP1 stability by direct interaction with YAP1

The oncogenic role of NF1 in gallbladder cancer through regulation of YAP1 stability by direct interaction with YAP1

  • J Transl Med. 2023 May 5;21(1):306. doi: 10.1186/s12967-023-04157-9.
Lingxiao Zhang # 1 2,  Lin Jiang # 1 3,  Ling Zeng # 1,  Zhaohui Jin 1,  Xuanjia Dong 1,  Yuhan Zhang 1,  Litian Chen 1,  Yijun Shu 4,  Yingbin Liu 5,  Ying Huang 6
Affiliations

Affiliations

  • 1 Department of General Surgery, Shanghai Key Laboratory of Biliary Tract Disease Research, Xinhua Hospital, Shanghai Jiao Tong University, Shanghai, 200092, China.
  • 2 Department of Biliary-Pancreatic Surgery, State Key Laboratory of Oncogenes and Related Genes, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China.
  • 3 School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China.
  • 4 Department of General Surgery, Shanghai Key Laboratory of Biliary Tract Disease Research, Xinhua Hospital, Shanghai Jiao Tong University, Shanghai, 200092, China. shuyijun@xinhuamed.com.cn.
  • 5 Department of Biliary-Pancreatic Surgery, State Key Laboratory of Oncogenes and Related Genes, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China. laoniulyb@163.com.
  • 6 Department of General Surgery, Shanghai Key Laboratory of Biliary Tract Disease Research, Xinhua Hospital, Shanghai Jiao Tong University, Shanghai, 200092, China. huangy@shsmu.edu.cn.
  • # Contributed equally.
PMID: 37147639 DOI: 10.1186/s12967-023-04157-9
Abstract

Background: Gallbladder Cancer (GBC) is the most prevalent and invasive biliary tract malignancy. As a GTPase-activating protein, Neurofibromin 1 (NF1) is a tumor suppressor that negatively regulates the Ras signaling pathway, and its abnormality leads to neurofibromatosis type 1 (NF-1) disease. However, the role of NF1 playing in GBC and the underlying molecular mechanism has not been defined yet.

Methods: A combination of NOZ and EH-GB1 cell lines as well as nude mice, were utilized in this study. mRNA expression and protein levels of NF1 and YAP1 were evaluated by quantitative Real-Time PCR (qRT-PCR), western blot (WB), and immunohistochemistry (IHC). In vitro and in vivo assays were performed to explore the biological effects of NF1 in NOZ and EH-GB1 cells via siRNA or lv-shRNA mediated knockdown. Direct interaction between NF1 and YAP1 was detected by confocal microscopy and co-immunoprecipitation (Co-IP), and further confirmed by GST pull-down assay and isothermal titration calorimetry assay (ITC). The stability of proteins was measured by western blot (WB) in the presence of cycloheximide.

Results: This study showed that a higher level of NF1 and YAP1 was found in GBC samples than in normal tissues and associated with worse prognoses. The NF1 knockdown impaired the proliferation and migration of NOZ in vivo and in vitro by downregulating YAP1 expression. Moreover, NF1 co-localized with YAP1 in NOZ and EH-GB1 cells, and the WW domains of YAP1 specifically recognized the PPQY motif of NF1. The structural modeling also indicated the hydrophobic interactions between YAP1 and NF1. On the other hand, YAP1 knockdown also impaired the proliferation of NOZ in vitro, phenocopying the effects of NF1 knockdown. Overexpression of YAP1 can partially rescue the impaired proliferation in NF1 stably knockdown cells. In mechanism, NF1 interacted with YAP1 and increased the stability of YAP1 by preventing ubiquitination.

Conclusions: Our findings discovered a novel oncogenic function of NF1 by directly interacting with YAP1 protein and stabilizing YAP1 to protect it from Proteasome degradation in NOZ cells. NF1 may serve as a potential therapeutic target in GBC.

Keywords

Gallbladder cancer (GBC); Hippo pathway; Neurofibromin 1 (NF1); YAP1.

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