Activation of protein kinase CB (PKCB) has been repeatedly implicated in tumor-induced angiogenesis. The PKCB-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses angiogenesis and was advanced for clinical development based upon this antiangiogenic activity. Activation of PKCB has now also been implicated in tumor cell proliferation, apoptosis, and tumor invasiveness. Herein, we show that Enzastaurin has a direct effect on human tumor cells, inducing apoptosis and suppressing the proliferation of cultured tumor cells. Enzastaurin treatment also suppresses the phosphorylation of GSK3B ser9 , ribosomal protein S6 S240/244 , and AKT Thr308 . Oral dosing with Enzastaurin to yield plasma concentrations similar to those achieved in clinical trials significantly suppresses the growth of human glioblastoma and colon carcinoma xenografts. As in cultured tumor cells, Enzastaurin treatment suppresses the phosphorylation of GSK3B in these xenograft tumor tissues. Enzastaurin treatment also suppresses GSK3B phosphorylation to a similar extent in peripheral blood mononuclear cells (PBMCs) from these treated mice. These data show that Enzastaurin has a direct antitumor effect and that Enzastaurin treatment suppresses GSK3B phosphorylation in both tumor tissue and in PBMCs, suggesting that GSK3B phosphorylation may serve as a reliable pharmacodynamic marker for Enzastaurin activity. With previously published reports, these data support the notion that Enzastaurin suppresses tumor growth through multiple mechanisms: direct suppression of tumor cell proliferation and the induction of tumor cell death coupled to the indirect effect of suppressing tumor-induced angiogenesis.

Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.

The eukaryotic translation initiation factor 4E (eIF4E) is frequently overexpressed in human cancers in relation to disease progression and drives cellular transformation, tumorigenesis, and metastatic progression in experimental models. Enhanced eIF4E function results from eIF4E overexpression and/or activation of the ras and phosphatidylinositol 3-kinase/AKT pathways and selectively increases the translation of key mRNAs involved in tumor growth, angiogenesis, and cell survival. Consequently, by simultaneously and selectively reducing the expression of numerous potent growth and survival factors critical for malignancy, targeting eIF4E for inhibition may provide an attractive therapy for many different tumor types. Recent work has now shown the plausibility of therapeutically targeting eIF4E and has resulted in the advance of the first eIF4E-specific therapy to clinical trials. These studies illustrate the increased susceptibility of tumor tissues to eIF4E inhibition and support the notion that the enhanced eIF4E function common to many tumor types may represent an Achilles' heel for cancer. [Cancer Res 2008;68(3):631-4] eIF4E Function and mRNA Discrimination Eukaryotic translation initiation factor 4E (eIF4E) binds the 5 ¶,7-methylguanosine cap structure of mRNAs, delivering these mRNAs to the eIF4F translation initiation complex, which is composed of eIF4E, the scaffolding protein eIF4G, and the ATPdependent RNA helicase eIF4A. The eIF4F complex then scans through the 5 ¶ untranslated region (UTR), unwinding mRNA secondary structure to expose the translation initiation codon and enable translation (1). eIF4F complex assembly is rate limiting for translation initiation and is largely dependent on eIF4E availability. Under normal cellular conditions, eIF4F complex assembly is limited as eIF4E is sequestered from eIF4G by binding to 4E binding proteins (4EBP). Stimulation of the phosphatidylinositol 3-kinase/AKT/mTOR pathway leads to hierarchical 4EBP phosphorylation, dislodging 4EBP from eIF4E and enabling assembly of the eIF4F complex (1).Enhanced eIF4F complex formation increases the translation of all cap-dependent mRNAs and thereby increases global protein synthesis rates. However, mRNAs vary widely in their inherent ''translatability, '' largely as a function of differences in the length and structure of their 5 ¶ UTRs. Cellular mRNAs most sensitive to alterations in eIF4E availability and eIF4F complex formation (i.e., weak mRNAs) have lengthy, G+C rich, highly structured 5 ¶ UTRs that encumber efficient RNA unwinding by eIF4F and subsequently prevent ribosome loading. Consequently, these mRNAs are most sensitive to eIF4E availability and are poorly translated under normal conditions when eIF4F complex formation is limiting. In contrast, most cellular mRNAs have relatively short, unstructured 5 ¶ UTRs (e.g., h-actin) that enable efficient scanning, initiation codon recognition, and ribosome loading and translation even when eIF4F complex levels are limiting. Whereas strong mRNAs (such as h-act...

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancers, particularly in more advanced cancers, suggesting that the AKT/protein kinase B (PKB) kinase, which is negatively regulated by PTEN, may be involved in human prostate cancer progression. We now show that AKT activation and activity are markedly increased in androgen-independent, prostate-specific antigen-positive prostate cancer cells (LNAI cells) established from xenograft tumors of the androgen-dependent LNCaP cell line. These LNAI cells show increased expression of integrin-linked kinase, which is putatively responsible for AKT activation/Ser-473 phosphorylation, as well as for increased phosphorylation of the AKT target protein, BAD. Furthermore, expression of the p27 Kip1 cell cycle regulator was diminished in LNAI cells, consistent with the notion that AKT directly inhibits AFX/Forkhead-mediated transcription of p27 Kip1 . To assess directly the impact of increased AKT activity on prostate cancer progression, an activated hAKT1 mutant was overexpressed in LNCaP cells, resulting in a 6-fold increase in xenograft tumor growth. Like LNAI cells, these transfectants showed dramatically reduced p27 Kip1 expression. Together, these data implicate increased AKT activity in prostate tumor progression and androgen independence and suggest that diminished p27Kip1 expression, which has been repeatedly associated with prostate cancer progression, may be a consequence of increased AKT activity.

SUMMARYFragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS pheno-types. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1 −/y), we show that phosphorylation of the mRNA 5′ cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1 −/y mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS.