Nicholas M. Dean

Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity

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.

Antisense oligonucleotide-based therapeutics for cancer

There has been steady progress in antisense technology over the past 14 years. We now have a far better appreciation of the attributes and limitations of the technology. Antisense oligonucleotides have been used to selectively inhibit thousands of genes in mammalian cells, hundreds, if not thousands, of genes in rodents and other species and multiple genes in humans. There are over 20 antisense drugs currently in clinical trials, several of which are showing promising results. Like any other class of drugs in development, there will continue to be successes and failures in the clinic. Despite some disappointments with the technology, it appears to be a valid platform for both drug discovery and as an experimental tool for functionalizing genes. Advances in the medicinal chemistry and formulation of antisense oligonucleotides will further enhance their therapeutic and commercial potential.

PKC-epsilon is required for mechano-sensitive activation of ERK1/2 in endothelial cells.

Mechano-sensitive regulation of endothelial cells (EC) function by shear stress is critical for flow-induced vasodilation and gene expression. Previous studies by our laboratory demonstrated that shear stress activates the 44- and 42-kDa extracellular signal-regulated kinases (ERK1/2) in EC in a time- and force-dependent manner. ERK1/2 activation was inhibited by protein kinase C (PKC) down-regulation with phorbol 12,13-dibutyrate (1 μm for 24 h) but not by calcium chelation with BAPTA-AM (acetoxymethyl ester of BAPTA) (75 μm for 30 min), suggesting that a novel PKC isoform (δ, ε, η, θ) mediates shear stress-induced ERK1/2 activation. Western blotting with PKC isoform-specific antibodies demonstrated expression of PKC-α, -ε, and -ζ isoforms in EC. PKC-ε was specifically inhibited by transfection with antisense PKC-ε phosphorothioate oligonucleotides (1,000 nm for 6 h). Antisense treatment decreased PKC-ε protein levels by 80 ± 13% after 72 h and completely inhibited shear stress-stimulated ERK1/2 activation. Scrambled PKC-ε oligonucleotides and antisense PKC-α and PKC-ζ oligonucleotides had no effect on ERK1/2 activity. PKC-ε appeared specific for mechano-sensitive ERK1/2 activation, as antisense PKC-ε oligonucleotides did not inhibit ERK1/2 activation by EGF or bradykinin but did inhibit ERK1/2 activation upon EC adhesion to fibronectin. These results define a pathway for shear stress-mediated ERK1/2 activation and establish a new function for PKC-ε as part of a mechano-sensitive signal transduction pathway in EC.

Inhibition of Bcl-xL expression sensitizes normal human keratinocytes and epithelial cells to apoptotic stimuli.

The epidermis is continually exposed to harmful mutagens that have the potential to cause DNA damage. To protect the skin from accumulating mutated cells, keratinocytes have developed a highly regulated mechanism of eliminating damaged cells through apoptosis. Bcl-xL is a well-described cell survival protein that when overexpressed in skin can protect keratinocytes from UV radiation-induced apoptosis. To begin to unravel the complex mechanisms that keratinocytes use to survive, we wanted to characterize the role of endogenous Bcl-xL in protecting cells from death. In this study, we describe the development and characterization of an antisense inhibitor to Bcl-xL. We show that this inhibitor reduces Bcl-xL RNA and protein in a concentration-dependent, sequence-specific manner. Furthermore, treatment of keratinocytes and epithelial cells with this inhibitor sensitizes these cells to UV-B radiation and cisplatinum treatment-induced apoptosis. Thus, these results offer direct evidence that Bcl-xL is critical in the protection of skin and epithelial cells from apoptosis and provide a basis for the role of Bcl-xL in keratinocyte and epithelial cell survival.

Selective down-regulation of glioma-associated oncogene 2 inhibits the proliferation of hepatocellular carcinoma cells.

The sonic hedgehog (Shh) pathway contributes to the initiation and progression of tumors with various origins when aberrantly activated. In this study, we investigated if the Shh pathway is important for the proliferation of hepatocellular carcinoma (HCC) cells and also began to identify which components of the pathway play a pivotal role in the biology of HCC. Expression levels of components in the pathway were measured, and glioma-associated oncogene (Gli) 2 levels were found to be considerably higher in human HCC lines compared with normal liver. Gli2 levels were also higher in tumor tissue from HCC patients compared with normal liver. Antisense oligonucleotides (ASO) were used to specifically down-regulate Gli2, and this led to decreased proliferation of various HCC cell lines. However, inhibition of Gli1 and Gli3 with ASOs did not decrease proliferation in most HCC cell lines and inhibitors targeting the upstream components of the pathway, including smoothened (Smo), displayed antiproliferative effects in only a subset of HCC cell lines. Moreover, in cancer cells harboring Smo mutations or unresponsive to the Smo inhibitor 3-keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine, the Gli2 ASO was still able to inhibit proliferation. The importance of Gli2 in HCC proliferation was further confirmed by the changes in expression levels of genes, such as Bcl-2, c-Myc, and p27, following suppression of Gli2 expression. Taken together, these results suggest that, among the Gli transcription factors, Gli2 plays a predominant role in the proliferation of HCC cells and the suppression of Gli2 expression may provide a useful therapeutic option for the treatment of HCC.

Use of antisense oligonucleotides in functional genomics and target validation.

With the completion of sequencing of the human genome, a great deal of interest has been shifted toward functional genomics-based research for identification of novel drug targets for treatment of various diseases. The major challenge facing the pharmaceutical industry is to identify disease-causing genes and elucidate additional roles for genes of known functions. Gene functionalization and target validation are probably the most important steps involved in identifying novel potential drug targets. This review focuses on recent advances in antisense technology and its use for rapid identification and validation of new drug targets. The significance and applicability of this technology as a beginning of the drug discovery process are underscored by relevant cell culture-based assays and positive correlation in specific animal disease models. Some of the antisense inhibitors used to validate gene targets are themselves being developed as drugs. The current clinical trials based on such leads that were identified in a very short time further substantiate the importance of antisense technology-based functional genomics as an integral part of target validation and drug target identification.

Inhibition of interleukin-1 type I receptor expression in human cell-lines by an antisense phosphorothioate oligodeoxynucleotide.

A 20 mer oligodeoxynucleotide designed to hybridize to specific 3-untranslated sequences in the type I receptor IL-1r mRNA was identified which inhibited the expression of both IL-1r mRNA and protein in human A549 lung carcinoma cells and human dermal fibroblasts. The oligodeoxynucleotide exhibited an IC50 value of approximately 100 nM in both cell lines and reduced IL-1r mRNA expression for up to 48 h. Multiple scrambled control oligonucleotides were without effect on IL-1r mRNA expression. Treatment of A549 cells with this oligodeoxynucleotide (but not scrambled controls) inhibited the IL-1 stimulated expression of the cell adhesion molecule ICAM-1 but was without effect on the TNF-alpha induction of this molecule. This study demonstrates that phosphorothioate oligodeoxynucleotides can selectively inhibit IL-1r expression leading to a reduction in IL-1 dependent gene expression.

Antisense methods for discrimination of phenotypic properties of closely related gene products: Jun kinase family.

Methods for the selection and characterization of antisense oligonucleotides for specifically eliminating closely related gene family members are available. High-throughput semiautomated methods using 96-well plate formats and array technology and improved assays are under active development that will streamline many steps and will likely merge. Second-generation 20-mer antisense phosphorothioate oligonucleotides containing 2-methoxyethyl groups at the first and last 6 nucleotides with improved nuclease resistance and RNA affinity are becoming available.