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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.
AuthorsArafath K Najumudeen, Itziar M D Posada, Benoit Lectez, Yong Zhou, Sebastian K-J Landor, Adyary Fallarero, Pia Vuorela, John Hancock, Daniel Abankwa
JournalBiochemistry (Biochemistry) Vol. 54 Issue 49 Pg. 7212-21 (Dec 15 2015) ISSN: 1520-4995 [Electronic] United States
PMID26568031 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
Chemical References
  • Hras protein, rat
  • Oncogene Proteins
  • Protein Synthesis Inhibitors
  • Extracellular Signal-Regulated MAP Kinases
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins
Topics
  • Amino Acid Substitution
  • Animals
  • Cell Membrane (genetics, metabolism, pathology)
  • Cell Transformation, Neoplastic (chemically induced, genetics, metabolism)
  • Cricetinae
  • Extracellular Signal-Regulated MAP Kinases (genetics, metabolism)
  • HEK293 Cells
  • Humans
  • MAP Kinase Signaling System (drug effects, genetics)
  • Mice
  • Mice, Knockout
  • Mutation, Missense
  • Neoplasms (chemically induced, genetics, metabolism, pathology)
  • Oncogene Proteins (genetics, metabolism)
  • PC12 Cells
  • Protein Synthesis Inhibitors (adverse effects, pharmacology)
  • Proto-Oncogene Proteins p21(ras) (genetics, metabolism)
  • Rats
  • ras Proteins (genetics, metabolism)

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