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

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.

DNMT3A Cooperates with YAP/TAZ to Drive Gallbladder Cancer Metastasis

“…Interestingly, YAP promotes the proliferation of GBC, [ 14 ] but we did not find any influence of DNMT3A on GBC growth. YAP and TAZ are the key downstream effectors of the Hippo pathway.…”

“…Although YAP/TAZ is required for metastasis in various solid tumors, [ 13 ] and the oncogenic role of YAP in the tumor growth of GBC has been confirmed, [ 14 ] the biological function of YAP/TAZ in driving GBC metastasis remains uncertain. We verified the biological validity of YAP/TAZ in GBC metastasis.…”

Glucose dysregulation promotes oncogenesis in human bladder cancer by regulating autophagy and YAP1/TAZ expression

“…Additionally, recent studies have demonstrated a close link between autophagy and Yes‐associated protein 1 (YAP1)‐WWTR1/ transcriptional coactivator with PDZ‐binding motif (TAZ) signalling. The carcinogenicity of YAP1/TAZ has been confirmed in many cancers 19–21 . After autophagy inhibition/induction, YAP1 /TAZ signalling increases or decreases owing to the regulation of CTNNA1/alpha‐catenin levels 22 .…”

“…The carcinogenicity of YAP1/TAZ has been confirmed in many cancers. 19 , 20 , 21 After autophagy inhibition/induction, YAP1 /TAZ signalling increases or decreases owing to the regulation of CTNNA1/alpha‐catenin levels. 22 In YAP1‐mutated phagocytic cells, the autophagic process was suppressed because of decreased YAP1‐TEAD transcriptional activity during Staphylococcus aureus infection.…”