ABSTRACT
The Akt activation inhibitor triciribine and the farnesyltransferase inhibitor tipifarnib have modest to little activity in clinical trials when used as single agents. In this article, preclinical data show that the combination is more effective than single agents both in cultured cells and in vivo. Combination index data analysis shows that this combination is highly synergistic at inhibiting anchorage-dependent growth of breast cancer cells. This synergistic interaction is also observed with structurally unrelated inhibitors of Akt (MK-2206) and farnesyltransferase (FTI-2153). The triciribine/tipifarnib synergistic effects are seen with several cancer cell lines including those from breast, leukemia, multiple myeloma and lung tumors with different genetic alterations such as K-Ras, B-Raf, PI3K (phosphoinositide 3-kinase), p53 and pRb mutations, PTEN, pRB and Ink4a deletions, and ErbB receptor overexpression. Furthermore, the combination is synergistic at inhibiting anchorage-independent growth and at inducing apoptosis in breast cancer cells. The combination is also more effective at inhibiting the Akt/mTOR/S6 kinase pathway. In an ErbB2-driven breast tumor transgenic mouse model, the combination, but not single agent, treatment with triciribine and tipifarnib induces significant breast tumor regression. Our findings warrant further investigation of the combination of farnesyltransferase and Akt inhibitors.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Carcinoma/drug therapy , Enzyme Inhibitors/administration & dosage , Farnesyltranstransferase/antagonists & inhibitors , Oncogene Protein v-akt/antagonists & inhibitors , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carcinoma/genetics , Carcinoma/pathology , Cell Line, Tumor , Disease Progression , Drug Evaluation, Preclinical , Drug Synergism , Enzyme Inhibitors/pharmacology , Female , Genes, erbB-2 , Humans , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Transgenic , Tumor Burden/drug effectsABSTRACT
The roles of eukaryotic DNA methylation in the repression of mRNA transcription and in the formation of heterochromatin have been extensively elucidated over the past several years. However, the role of DNA methylation in transcriptional activation remains a mystery. In particular, it is not known whether the transcriptional activation of methylated DNA is promoter-specific, depends directly on sequence-specific DNA-binding proteins, or is facilitated by the methylation. Here we report that the sequence-specific DNA-binding protein, RFX, previously shown to mediate the transition from an inactive to an active chromatin structure, activates a methylated promoter. RFX is capable of mediating enhanceosome formation on a methylated promoter, thereby mediating a transition from a methylation-dependent repression of the promoter to a methylation-dependent activation of the promoter. These results indicate novel roles for DNA methylation and sequence-specific DNA-binding proteins in transcriptional activation.
Subject(s)
DNA-Binding Proteins/metabolism , HLA-DR Antigens/genetics , Promoter Regions, Genetic , Transcription Factors/metabolism , B-Lymphocytes/metabolism , Base Sequence , Binding Sites/genetics , Cell Line , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Methylation , Gene Expression Regulation , HLA-DR alpha-Chains , Humans , Models, Biological , Molecular Sequence Data , Octamer Transcription Factor-1/metabolism , Regulatory Factor X Transcription Factors , Transfection , YY1 Transcription Factor/metabolismABSTRACT
Stat3 protein has an important role in oncogenesis and is a promising anticancer target. Indirubin, the active component of a traditional Chinese herbal medicine, has been shown previously to inhibit cyclin-dependent kinases, resulting in cell cycle arrest. Here, we show that the indirubin derivatives E564, E728, and E804 potently block constitutive Stat3 signaling in human breast and prostate cancer cells. In addition, E804 directly inhibits Src kinase activity (IC(50) = 0.43 microM) in an in vitro kinase assay. Levels of tyrosyl phosphorylation of c-Src are also reduced in cultured cells 30 min after E804 treatment. Tyrosyl phosphorylation of Stat3, which is known to be phosphorylated by c-Src, was decreased, and constitutive Stat3 DNA binding-activity was suppressed in cells 30 min after E804 treatment. The antiapoptotic proteins Mcl-1 and Survivin, which are encoded in target genes of Stat3, were down-regulated by indirubin derivatives, followed by induction of apoptosis. These results demonstrate that E804 directly blocks the Src-Stat3 signaling pathway, suggesting that the antitumor activity of indirubin compounds is at least partially due to inhibition of this pathway.
Subject(s)
Apoptosis/drug effects , DNA-Binding Proteins/antagonists & inhibitors , Indoles/pharmacology , Trans-Activators/antagonists & inhibitors , Breast Neoplasms , Cell Cycle/drug effects , Cell Line, Tumor , Female , Growth Inhibitors/pharmacology , Humans , Inhibitor of Apoptosis Proteins , Kinetics , Male , Microtubule-Associated Proteins/drug effects , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins , Phosphorylation , Prostatic Neoplasms , STAT3 Transcription Factor , Signal Transduction/drug effects , Signal Transduction/physiology , SurvivinABSTRACT
Akt/protein kinase B is a major cell survival pathway through phosphorylation of proapoptotic proteins Bad and Bax and of additional apoptotic pathways linked to Forkhead proteins glycogen synthase kinase-3beta and ASK1. To further explore the mechanism by which Akt regulates cell survival, we identified an Akt interaction protein by yeast two-hybrid screening. It is highly homologous to ARG-binding protein 2 (ArgBP2) with splicing exon 8 of the coding region of the ArgBP2. As two splicing isoforms (ArgBP2alpha and -beta) of ArgBP2 have been identified (Wang, B., Golemis, E. A., and Kruh, G. D. (1997) J. Biol. Chem. 272, 17542-17550), it was named ArgBP2gamma. ArgBP2gamma contains four Akt phosphorylation consensus sites, a SoHo motif, and three Src homology (SH) 3 domains and binds to C-terminal proline-rich motifs of Akt through its first and second SH3 domains. It also interacts with p21-activated protein kinase (PAK1) via its first and third SH3 domains, indicating the SH3 domains of ArgBP2gamma as docking sites for Akt and PAK1. Akt phosphorylates ArgBP2gamma in vitro and in vivo. Expression of ArgBP2gamma induces PAK1 activity and overrides apoptosis induced by ectopic expression of Bad or DNA damage. Nonphosphorylatable ArgBP2gamma-4A and SH3 domain-truncated mutant ArgBP2gamma inhibit Akt-induced PAK1 activation and reduce Akt and PAK1 phosphorylation of Bad and antiapoptotic function. These data indicate that ArgBP2gamma is a physiological substrate of Akt, functions as an adaptor for Akt and PAK1, and plays a role in Akt/PAK1 cell survival pathway.