Author of

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  MO15 - Novel Genes and Pathways (ID 89)

  • Event: WCLC 2013
  • Type: Mini Oral Abstract Session
  • Track: Biology
  • Presentations: 1
  • Moderators:Y. Ohe, G. Reid
  • Coordinates: 10/29/2013, 16:15 - 17:45, Parkside Ballroom A, Level 1

  • 12214

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    MO15.03 - Deciphering the RASSF1A signaling pathway in lung cancer cells reveals a metastasis-suppressor role through YAP-dependent epithelial-mesenchymal transition (EMT) (ID 3189)

    16:25 - 16:30  |  Author(s): J. Camonis
    • Abstract
    • Presentation
    • Slides

    Background
    RASSF1A gene promoter hypermethylation was previously shown to predict poor overall survival in the IFCT-0002 randomized phase 3 trial of neo-adjuvant platinum-based chemotherapy, in early stage (I & II) NSCLC. We investigated the molecular and cellular basis for such a dramatic influence.

    Methods
    We studied isogenic immortalized bronchial, non-tumorogenic, HBEC3 cell lines only differing by their K-Ras status (wild-type or mutant K-Ras Val12 allele), and a panel of lung cancer cell lines recapitulating the main molecular alterations encountered in lung cancer. RASSF1A protein was depleted by 80% using 2 specific siRNAs, followed by the evaluation of EMT markers and cell motility regualors using qRT-PCR, Western blot or Immunofluorescence. Migration of transfected cells was assayed by 2D wound-healing migration assays or 3D migration assays using transwell devices with or without a matrigel coating mimicking basement membrane (invasion assay), or an endothelial cell monolayer (trans-endothelial cell invasion). Phenotypic rescue was studied by using plasmids encoding full-length RASSF1A or RASSF1C isoform, and a construct encoding a SARAH-deleted RASSF1A protein, unable to interact with the Hippo/MST kinase. We also tested co-transfection of RASSF1A siRNAs together with siRNAs directed against Hippo pathway members LATS1/2, WW45, YAP. Depletion of RASSF1A was finally combined with expression of wild-type, activated or dominant negative RhoA, RhoB, Rac1 or CDC42 constructs.

    Results
    In each bronchial/lung cancer cell line tested, RASSF1A silencing led to EMT resulting in E-cadherin, Syndecan1, Zo-1, miR200 decrease and concurrent N-cadherin, vimentin, Twist1, miR-21 increase. RASSF1A silencing-induced EMT was associated with cytoplasmic to nucleus translocation of YAP transcription factor, the terminal effector of the Hippo signaling pathway. RASSF1A silencing reduced cell adhesion and increased 2D cell motility with collective migration features. RASSF1A knock-down increased 3D migration, invasion as trans-endothelial migration. These effects correlated with the up-regulation of RhoA, RhoC, CDC42, MMP2/14 mRNAs and down-regulation of RhoB, DIA1 and MMP9 mRNAs. We also observed an increase of adhesion/invasion signaling proteins, i.e. CD44v6, cofilin, ERM and NF2, cofilin being activated by inhibition of LIMK-induced phosphorylation. Finally we report that immortalized non-tumorogenic cell lines, unable to grow without adhesion, acquired the capacity to grow in soft agar when RASSFIA was knocked-down. Those effects were rescued by co-transfection of RASSF1A siRNAs with full-length RASSF1A cDNA, showing the specificity of the motile phenotype induced by RASSF1A silencing, but not by RASSF1C nor SARAH-deficient RASSF1A plasmids. SiRASSF1A-induced cell migration was inhibited by LATS1/LATS2/WW45 or YAP siRNAs, showing the involvement of the Lats/YAP signaling cascade. We finally show that RASSF1A knockdown-promoted migration was inhibited by using RhoB-Val14 constitutively active cDNA but not RhoBN19 dominant negative construct, and by specific RhoB GEFs or RhoB effectors (DIA1) constructs.

    Conclusion
    In lung cells, the RASSF1A protein acts as a migration-suppressor protein by regulating the LIMK/cofilin pathway through RhoB signaling. RASSF1A prevents YAP induced EMT by inhibiting its nuclear accumulation through LATS1/2 signaling, whereas Hippo/MST kinase seemed dispensable. We thus provide evidence, for the first time in human lung cancer cells, for a direct connection between RASSF1A signaling and the LATS/YAP pathway.

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