Journal
BIOCHEMISTRY
Volume 54, Issue 49, Pages 7212-7221Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.5b00724
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Funding
- Academy of Finland
- Sigrid Juselius Foundation
- Cancer Society of Finland
- Marie-Curie Reintegration Grant
- Jane and Aatos Erkko Foundation
- Texas Digestive Diseases Center Pilot and Feasibility grant [P30 DK56338]
- CPRIT (Cancer Prevention Research Institute of Texas) [RP130059]
- ISB graduate school
- Drug Discovery and Chemical Biology (DDCB) network of Biocenter Finland
- NordForsk Nordic Chemical Biology network
- Swedish Cultural Foundation of Finland
- Magnus Ehrnrooth Foundation
- Waldemar von Frenckell Foundation
- K. Albin Johansson Foundation
- Knut and Alice Wallenberg Foundation
- Cancer Foundation Finland sr [100073] Funding Source: researchfish
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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 nanodusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanodusters 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-nanodustering 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, cydoheximide 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 cydoheximide 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.
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