Cancer; Differentiation; Flow cytometry; Inhibitors; RASopathy; Ras; Protein Isoforms; ras Proteins; ras Proteins/metabolism; ras Proteins/genetics; Cell Line; Humans; Cell Differentiation; Mutation; Protein Isoforms/metabolism; Protein Isoforms/genetics; Cell Biology
Abstract :
[en] The RAS-MAPK-pathway is aberrantly regulated in cancer and developmental diseases called RASopathies. While typically the impact of Ras on the proliferation of various cancer cell lines is assessed, it is poorly established how Ras affects cellular differentiation. Here we implement the C2C12 myoblast cell line to systematically study the effect of Ras mutants and Ras-pathway drugs on differentiation. We first provide evidence that a minor pool of Pax7+ progenitors replenishes a major pool of transit amplifying cells that are ready to differentiate. Our data indicate that Ras isoforms have distinct roles in the differentiating culture, where K-Ras depletion increases and H-Ras depletion decreases terminal differentiation. This assay could therefore provide significant new insights into Ras biology and Ras-driven diseases. In line with this, we found that all oncogenic Ras mutants block terminal differentiation of transit amplifying cells. By contrast, RASopathy associated K-Ras variants were less able to block differentiation. Profiling of eight targeted Ras-pathway drugs on seven oncogenic Ras mutants revealed their allele-specific activities and distinct abilities to restore normal differentiation as compared to triggering cell death. In particular, the MEK-inhibitor trametinib could broadly restore differentiation, while the mTOR-inhibitor rapamycin broadly suppressed differentiation. We expect that this quantitative assessment of the impact of Ras-pathway mutants and drugs on cellular differentiation has great potential to complement cancer cell proliferation data.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
CHIPPALKATTI, Rohan ✱; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
PARISI, Bianca ✱; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
Kouzi, Farah; Cancer Cell Biology and Drug Discovery group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette 4362, Luxembourg
LAURINI, Christina ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
Ben Fredj, Nesrine; Cancer Cell Biology and Drug Discovery group, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette 4362, Luxembourg
ABANKWA, Daniel ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
✱ These authors have contributed equally to this work.
External co-authors :
no
Language :
English
Title :
RAS isoform specific activities are disrupted by disease associated mutations during cell differentiation.
This work was supported by funds provided by the University of Luxembourg and a grant from the Luxembourg National Research Fund (FNR) grant C19/BM/13673303-PolaRAS2 to D.K.A.
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