title: Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.
abstract: Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.
authors.affiliation: Turku Centre for Biotechnology, Åbo Akademi University , Tykistökatu 6B, 20520 Turku, Finland.
authors.forename: Arafath K
authors.initials: AK
authors.lastname: Najumudeen
authors.affiliation: Turku Centre for Biotechnology, Åbo Akademi University , Tykistökatu 6B, 20520 Turku, Finland.
authors.forename: Itziar M D
authors.initials: IM
authors.lastname: Posada
authors.affiliation: Turku Centre for Biotechnology, Åbo Akademi University , Tykistökatu 6B, 20520 Turku, Finland.
authors.forename: Benoit
authors.initials: B
authors.lastname: Lectez
authors.affiliation: University of Texas Health Science Center at Houston , Medical School, Houston, Texas 77030, United States.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Turku Centre for Biotechnology, Åbo Akademi University , Tykistökatu 6B, 20520 Turku, Finland.|Department of Cell and Molecular Biology (CMB), Karolinska Institutet , SE-171 77 Stockholm, Sweden.
authors.forename: Sebastian K-J
authors.initials: SK
authors.lastname: Landor
authors.affiliation: Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki , FI-00014 Helsinki, Finland.
authors.forename: Adyary
authors.initials: A
authors.lastname: Fallarero
authors.affiliation: Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki , FI-00014 Helsinki, Finland.
authors.forename: Pia
authors.initials: P
authors.lastname: Vuorela
authors.affiliation: University of Texas Health Science Center at Houston , Medical School, Houston, Texas 77030, United States.
authors.forename: John
authors.initials: J
authors.lastname: Hancock
authors.affiliation: Turku Centre for Biotechnology, Åbo Akademi University , Tykistökatu 6B, 20520 Turku, Finland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Abankwa
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced* / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D020125:Mutation, Missense; D009369:Neoplasms / Q000139:chemically induced* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D011500:Protein Synthesis Inhibitors / Q000009:adverse effects* / Q000494:pharmacology; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C528429:Hras protein, rat; D015513:Oncogene Proteins; D011500:Protein Synthesis Inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins