Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells.
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 metalloproteinases 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 (RafDeltaN). 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.
Kato-Stankiewicz
Juran
J
Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095-1489, USA.
Hakimi
Irina
I
Zhi
Gang
G
Zhang
Jie
J
Serebriiskii
Ilya
I
Guo
Lea
L
Edamatsu
Hironori
H
Koide
Hiroshi
H
Menon
Sanjay
S
Eckl
Robert
R
Sakamuri
Sukumar
S
Lu
Yingchun
Y
Chen
Quin-Zene
QZ
Agarwal
Seema
S
Baumbach
William R
WR
Golemis
Erica A
EA
Tamanoi
Fuyuhiko
F
Khazak
Vladimir
V
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D015723:Gene Library; D006801:Humans; D008156:Luciferases; D020935:MAP Kinase Signaling System; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics*; D027602:Serum Response Element; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*; D014170:Transformation, Genetic; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D018808:Transcription Factor AP-1; D008156:Luciferases; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins