American Association for Cancer Research
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00085472can151008-sup-148249_3_supp_3272407_nzgdd1.pptx (2.22 MB)

Figure S6. sh-mediated RASSF1A depletion in HBEC-3 cells. from RASSF1A Suppresses the Invasion and Metastatic Potential of Human Non–Small Cell Lung Cancer Cells by Inhibiting YAP Activation through the GEF-H1/RhoB Pathway

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posted on 2023-03-30, 23:52 authored by Fatéméh Dubois, Maureen Keller, Olivier Calvayrac, Fabrice Soncin, Lily Hoa, Alexander Hergovich, Maria-Carla Parrini, Julien Mazières, Mélissa Vaisse-Lesteven, Jacques Camonis, Guénaëlle Levallet, Gérard Zalcman

HBEC-3 cells were transfected with non-silencing shRNA (Control), shRASSF1A-777 and shRASSF1A-779. A) Changes in RASSF1A and Yap expression assessed by immunofluorescence (Red=RASSF1A, Green=Yap, Blue=DAPI) experiments. Scale bar represents 50 µm. B) The migration speed (µm/h) of transfected cells was determined by wound assay on collagen IV. Mitomycin C (1 µg/ml) was added to medium 12h before wounding. The invasion capacity was assessed using a BioCoat Matrigel Invasion Chamber. The relative invasion was normalized to that of the cells transfected with Control. C) Representative images of colony formation of transfected cells in soft agar. Scale bar represents 20 µm. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p<0.05, **p<0.01 and ***p<0.001, using an ANOVA test followed by Dunnett's test.

Funding

Cancéropole Nord-Ouest

Ligue contre le Cancer de Normandie

CNMR

Wellcome Trust

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ARTICLE ABSTRACT

Inactivation of the tumor suppressor gene RASSF1A by promoter hypermethylation represents a key event underlying the initiation and progression of lung cancer. RASSF1A inactivation is also associated with poor prognosis and may promote metastatic spread. In this study, we investigated how RASSF1A inactivation conferred invasive phenotypes to human bronchial cells. RNAi-mediated silencing of RASSF1A induced epithelial-to-mesenchymal transition (EMT), fomenting a motile and invasive cellular phenotype in vitro and increased metastatic prowess in vivo. Mechanistic investigations revealed that RASSF1A blocked tumor growth by stimulating cofilin/PP2A–mediated dephosphorylation of the guanine nucleotide exchange factor GEF-H1, thereby stimulating its ability to activate the antimetastatic small GTPase RhoB. Furthermore, RASSF1A reduced nuclear accumulation of the Hippo pathway transcriptional cofactor Yes-associated protein (YAP), which was reinforced by RhoB activation. Collectively, our results indicated that RASSF1 acts to restrict EMT and invasion by indirectly controlling YAP nuclear shuttling and activation through a RhoB-regulated cytoskeletal remodeling process, with potential implications to delay the progression of RASSF1-hypermethylated lung tumors. Cancer Res; 76(6); 1627–40. ©2016 AACR.

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