Cyclin D3 is down-regulated by rapamycin in HER-2-overexpressing breast cancer cells.
Rapamycin and its analogues are being tested as new antitumor agents. Rapamycin binds to FKBP-12 and this complex inhibits the activity of FRAP/mammalian target of rapamycin, which leads to dephosphorylation of 4EBP1 and p70 S6 kinase, resulting in blockade of translation initiation. We have found that RAP inhibits the growth of HER-2-overexpressing breast cancer cells. The phosphorylation of mammalian target of rapamycin, p70 S6 kinase, and 4EBP1 is inhibited by rapamycin and cells are arrested in the G1 phase, as determined by growth assays, fluorescence-activated cell sorting analysis, and bromodeoxyuridine incorporation studies. Rapamycin causes down-regulation of cyclin D3 protein, retinoblastoma hypophosphorylation, loss of cyclin-dependent kinase (cdk) 4, cdk6, and cdk2 activity. The half-life of cyclin D3 protein decreases after rapamycin treatment, but not its synthesis, whereas the synthesis or half-life of cyclin D1 protein is not affected by the drug. Additionally, rapamycin caused accumulation of ubiquitinated forms of cyclin D3 protein, proteasome inhibitors blocked the effect of rapamycin on cyclin D3, and rapamycin stimulated the activity of the proteasome, showing that the effect of rapamycin on cyclin D3 is proteasome proteolysis dependent. This effect depends on the activity of HER-2 because Herceptin, a neutralizing antibody against HER-2, is able to block both the induction of proteasome activity and the cyclin D3 down-regulation due to rapamycin. Furthermore, inhibition of HER-2 gene expression by using small interfering RNA blocked the rapamycin effects on cyclin D3. These data indicate that rapamycin causes a G1 arrest in HER-2-overexpressing breast cancer cells that is associated with a differential destabilization and subsequent down-regulation of cyclin D3 protein.
García-Morales
Pilar
P
Instituto de Biología Molecular y Celular, Edificio Torregaitán, Universidad Miguel Hernández, 03202 Elche, Spain.
Hernando
Eva
E
Carrasco-García
Estefanía
E
Menéndez-Gutierrez
María Piedad
MP
Saceda
Miguel
M
Martínez-Lacaci
Isabel
I
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D056743:Cyclin D3; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics*; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D051058:Oncogene Protein v-akt; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D061988:Proteasome Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D012175:Retinoblastoma / Q000235:genetics; D020123:Sirolimus / Q000276:immunology / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases; D014452:Ubiquitins / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D056743:Cyclin D3; D051357:Cyclin-Dependent Kinase 2; D016213:Cyclins; D051058:Oncogene Protein v-akt; D019869:Phosphatidylinositol 3-Kinases; D061988:Proteasome Inhibitors; D011494:Protein Kinases; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D014452:Ubiquitins; C534696:CCND3 protein, human; C546842:MTOR protein, human