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.

Rapalogs can promote cancer cell stemness in vitro in a Galectin-1 and H-ras-dependent manner

“…As previously observed with CHX [ 23 ], rapalogs significantly increased MDA-MB-231 mammosphere formation in a H-ras dependent manner (Figure 2A , 2B ). Note, that H-ras knockdown alone increased sphere formation relative to the scramble control, consistent with its negative effect on K-ras nanoscale organization and signaling, which is mediated by perturbation of the phosphatidylserine distribution by oncogenic H-ras [ 24 ].…”

“…We recently found that the commonly used protein synthesis inhibitor cycloheximide (CHX), was paradoxically able to increase the number of mammospheres and tumor growth in a H-ras dependent manner [ 23 ]. CHX exhibits a typically neglected polypharmacology, as it is not only an inhibitor of protein synthesis, but also of FKBP12, which in complex with rapalogs inhibits mTORC1.…”

“…Accordingly, expression of GFP-H-rasG12V or K-rasG12V induced differentiation of PC12 cells, as can be seen by neurite formation (Figure 1G ). Previously, others and we have shown that H-rasG12V-driven PC12 cell differentiation was increased by treatments that increased H-ras nanoclustering, such as Gal-1 expression [ 28 ] or CHX treatment [ 23 ]. In line with the H-ras-specific effect of rapamycin on nanoclustering upon rapamycin treatment (Figure 1C , 1D ), only H-rasG12V transfected PC12 cells showed an increased differentiation with rapamycin as compared to the non-treated control, while K-rasG12V transfected PC12 cells did not (Figure 1G ).…”

Opposite feedback from mTORC1 to H-ras and K-ras4B downstream of SREBP1

“…1B ). Unlike CHX 38 , the other PSI robustly blocked mammosphere formation of MDA-MB-231 cells, as would be expected from the inhibition of cellular protein synthesis (Supplementary Fig. 1C ).…”

“…We therefore followed up on our recent findings, showing that the protein synthesis inhibitor cycloheximide (CHX) specifically increases H-ras nanoclustering (i.e. nanoscale membrane signalling complexes of Ras 37 ) and downstream signalling, whereby it paradoxically promoted mammosphere growth in a H-ras-dependent manner 38 .…”