Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells

摘要

The interaction of activated Ras with Raf initiates signaling cascades that contribute to a significant percentage of human tumors, suggesting that agents that specifically disrupt this interaction might have desirable chemotherapeutic properties. We used a subtractive forward two-hybrid approach to identify small molecule compounds that block the interaction of Ras with Raf. These compounds (MCP1 and its derivatives, 53 and 110) reduced serum-induced transcriptional activation of serum response element as well as Ras-induced transcription by way of the AP-1 site. They also inhibited Ras-induced Raf-1 activation in human embryonic kidney 293 cells, Raf-1 and mitogen-activated protein kinase kinase 1 activities in HT1080 fibrosarcoma cells, and epidermal growth factor-induced Raf-1 activation in A549 lung carcinoma cells. The MCP compounds caused reversion of ras-transformed phenotypes including morphology, in vitro invasiveness, and anchorage-independent growth of HT1080 cells. Decreased level of matrix metal-loproteinases was also observed. Further characterization showed that MCP compounds restore actin stress fibers and cause flat reversion in NIH 3T3 cells transformed with H-Ras (V12) but not in NIH 3T3 cells transformed with constitutively active Raf-1 (Raf ΔN). Finally, we show that MCP compounds inhibit anchorage-independent growth of A549 and PANC-1 cells harboring K-ras mutation. Furthermore, MCP110 caused G_1 enrichment of A549 cells with the decrease of cyclin D level. These results highlight potent and specific effects of MCP compounds on cancer cells with intrinsic Ras activation.