Tumor cell-targeted delivery of nanoconjugated oligonucleotides in composite spheroids.

Standard tissue culture has often been a poor model for predicting the efficacy of anti-cancer agents including oligonucleotides. In contrast to the simplicity of monolayer tissue cultures, a tumor mass includes tightly packed tumor cells, tortuous blood vessels, high levels of extracellular matrix, and stromal cells that support the tumor. These complexities pose a challenge for delivering therapeutic agents throughout the tumor, with many drugs limited to cells proximal to the vasculature. Multicellular tumor spheroids are superior to traditional monolayer cell culture for the assessment of cancer drug delivery, since they possess many of the characteristics of metastatic tumor foci. However, homogeneous spheroids comprised solely of tumor cells do not account for some of the key aspects of metastatic tumors, particularly the interaction with host cells such as fibroblasts. Further, homogeneous culture does not allow for the assessment of targeted delivery to tumor versus host cells. Here we have evaluated delivery of targeted and untargeted oligonucleotide nanoconjugates and of oligonucleotide polyplexes in both homogeneous and composite tumor spheroids. We find that inclusion of fibroblasts in the spheroids reduces delivery efficacy of the polyplexes. In contrast, targeted multivalent RGD-oligonucleotide nanoconjugates were able to effectively discriminate between melanoma cells and fibroblasts, thus providing tumor-selective uptake and pharmacological effects.

Multivalent cyclic RGD conjugates for targeted delivery of small interfering RNA.

We have designed, synthesized, and tested conjugates of chemically modified luciferase siRNA (Luc-siRNA) with bi-, tri-, and tetravalent cyclic(arginine-glycine-aspartic) (cRGD) peptides that selectively bind to the αvß3 integrin. The cellular uptake, subcellular distribution, and pharmacological effects of the cRGD-conjugated Luc-siRNAs compared to those of unconjugated controls were examined using a luciferase reporter cassette stably transfected into αvß3 positive M21(+) human melanoma cells. The M21(+) cells exhibited receptor-mediated uptake of cRGD-siRNA conjugates but not of unconjugated control siRNA. The fluorophore-tagged cRGD-siRNA conjugates were taken up by a caveolar endocytotic route and primarily accumulated in cytosolic vesicles. The bi-, tri-, and tetravalent cRGD conjugates were taken up by M21(+) cells to approximately the same degree. However, there were notable differences in their pharmacological effectiveness. The tri- and tetravalent versions produced progressive, dose-dependent reductions in the level of luciferase expression, while the bivalent version had little effect. The basis for this divergence of uptake and effect is currently unclear. Nonetheless, the high selectivity and substantial "knock down" effects of the multivalent cRGD-siRNA conjugates suggest that this targeting and delivery strategy deserves further exploration.

Integrin targeted delivery of gene therapeutics.

Integrins have become key targets for molecular imaging and for selective delivery of anti-cancer agents. Here we review recent work concerning the targeted delivery of antisense and siRNA oligonucleotides via integrins. A variety of approaches have been used to link oligonucleotides to ligands capable of binding integrins with high specificity and affinity. This includes direct chemical conjugation, incorporating oligonucleotides into lipoplexes, and use of various polymeric nanocarriers including dendrimers. The ligand-oligonucleotide conjugate or complex associates selectively with the integrin, followed by internalization into endosomes and trafficking through subcellular compartments. Escape of antisense or siRNA from the endosome to the cytosol and nucleus may come about through endogenous trafficking mechanisms, or because of membrane disrupting capabilities built into the conjugate or complex. Thus a variety of useful strategies are available for using integrins to enhance the pharmacological efficacy of therapeutic oligonucleotides.

The Carolina Center of Cancer Nanotechnology Excellence: past accomplishments and future perspectives.

The Carolina Center of Cancer Nanotechnology Excellence (C-CCNE) is funded by the National Cancer Institute and is based at the University of North Carolina. The C-CCNE features interactions between physical and biological scientists in a series of projects and cores that work together to quickly harness innovations in nanotechnology for the early diagnosis and treatment of cancer. Two key focus areas of the C-CCNE are, first, the selective delivery of drugs and imaging agents utilizing advanced nanoparticle technology, and second, novel approaches to imaging and radiotherapy utilizing carbon nanotube-based x-ray sources.

Biological effects of hexitol and altritol-modified siRNAs targeting B-Raf.

Increasing the effectiveness of siRNAs through chemical modification is an important task. Here we describe altritol and hexitol modified oligonucleotides targeting the B-Raf oncogene that is critical for the growth and survival of melanoma cells. Using assays for apoptosis, DNA synthesis, colony formation and B-Raf protein and message levels, we demonstrate that certain hexitol modifications can improve the effectiveness of B-Raf siRNAs and also increase duration of action. Altritol modified siRNAs were similar to or slightly less effective than unmodified B-Raf siRNA. Modifications at the 3' or 5' end of the sense strand, at the 3' end of the antisense strand, or within either strand were well tolerated. The basis for the increased effectiveness of the hexitol-modified siRNAs is not fully understood but may be partly due to increased stability to nucleases.

Cellular delivery and biological activity of antisense oligonucleotides conjugated to a targeted protein carrier.

Targeted delivery can potentially improve the pharmacological effects of antisense and siRNA oligonucleotides. Here, we describe a novel bioconjugation approach to the delivery of splice-shifting antisense oligonucleotides (SSOs). The SSOs are linked to albumin via reversible S-S bonds. The albumin is also conjugated with poly(ethylene glycol) (PEG) chains that terminate in an RGD ligand that selectively binds the alphavbeta3 integrin. As a test system, we utilized human melanoma cells that express the alphavbeta3 integrin and that also contain a luciferase reporter gene that can be induced by delivery of SSOs to the cell nucleus. The RGD-PEG-SSO-albumin conjugates were endocytosed by the cells in an RGD-dependent manner; using confocal fluorescence microscopy, evidence was obtained that the SSOs accumulate in the nucleus. The conjugates were able to robustly induce luciferase expression at concentrations in the 25-200 nM range. At these levels, little short-term or long-term toxicity was observed. Thus, the RGD-PEG-albumin conjugates may provide an effective tool for targeted delivery of oligonucleotides to certain cells and tissues.

Effects of structure of Rho GTPase-activating protein DLC-1 on cell morphology and migration.

DLC-1 encodes a Rho GTPase-activating protein (RhoGAP) and negative regulator of specific Rho family proteins (RhoA-C and Cdc42). DLC-1 is a multi-domain protein, with the RhoGAP catalytic domain flanked by an amino-terminal sterile alpha motif (SAM) and a carboxyl-terminal START domain. The roles of these domains in the regulation of DLC-1 function remain to be determined. We undertook a structure-function analysis involving truncation and missense mutants of DLC-1. We determined that the amino-terminal SAM domain functions as an autoinhibitory domain of intrinsic RhoGAP activity. Additionally, we determined that the SAM and START domains are dispensable for DLC-1 association with focal adhesions. We then characterized several mutants for their ability to regulate cell migration and identified constitutively activated and dominant negative mutants of DLC-1. We report that DLC-1 activation profoundly alters cell morphology, enhances protrusive activity, and can increase the velocity but reduce directionality of cell migration. Conversely, the expression of the amino-terminal domain of DLC-1 acts as a dominant negative and profoundly inhibits cell migration by displacing endogenous DLC-1 from focal adhesions.

Selective killing of Smad4-negative tumor cells via a designed repressor strategy.

Smad4 is a key tumor suppressor that is frequently deleted or inactive in pancreatic and colon tumors. In this report, we describe an approach for attaining selective killing of Smad4-deficient tumor cells. Using a vector system involving a designed repressor with zinc finger binding domains and the herpes simplex virus thymidine kinase (HSV-TK) "suicide gene," we demonstrate Smad4-responsive regulation of HSV-TK expression and consequent altered susceptibility to the prodrug ganciclovir (GCV). In pancreatic tumor cell lines stably transfected with the vector system, a robust differential of HSV-TK expression and GCV toxicity was attained depending on the presence or absence of cotransfected Smad4. In matched colon tumor cell lines lacking Smad4 or expressing physiological levels of Smad4, an adenoviral version of the vector system attained a significant degree of preferential killing of Smad4-negative tumor cells in response to GCV. These findings demonstrate the possibility of achieving selective killing of pancreatic and colon cells depending on their Smad4 status.

Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis.

We describe the synthesis and characterization of a 5' conjugate between a 2'-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine-glycine-aspartic acid) peptide that is a high-affinity ligand for the alphavbeta3 integrin. We used alphavbeta3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells. The RGD-623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while 'free' 623 did not. However, the kinetics of luciferase expression was distinct; the RGD-623 conjugate produced a gradual increase followed by a gradual decline, while the cationic lipid-623 complex caused a rapid increase followed by a monotonic decline. The subcellular distribution of the oligonucleotide delivered using cationic lipids included both cytoplasmic vesicles and the nucleus, while the RGD-623 conjugate was primarily found in cytoplasmic vesicles that partially co-localized with a marker for caveolae. Both the cellular uptake and the biological effect of the RGD-623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptide-oligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the alphavbeta3 integrin.

Clinging to life: cell to matrix adhesion and cell survival.

Cell to matrix adhesion regulates cellular homeostasis in multiple ways. Integrin attachment to the extracellular matrix mediates this regulation through direct and indirect connections to the actin cytoskeleton, growth factor receptors, and intracellular signal transduction cascades. Disruption of this connection to the extracellular matrix has deleterious effects on cell survival. It leads to a specific type of apoptosis known as anoikis in most non-transformed cell types. Anchorage independent growth is a critical step in the tumorigenic transformation of cells. Thus, breaching the anoikis barrier disrupts the cell's defenses against transformation. This review examines recent investigations into the molecular mechanisms of anoikis to illustrate current understanding of this important process.

Regulation of p21-activated kinase-independent Rac1 signal transduction by nischarin.

Nischarin regulates Rac1-dependent cell motility by interaction with and inhibition of the p21-activated kinase (PAK1). In addition to regulating the activation of PAK1, Rac1 controls multiple downstream pathways to regulate cell growth and differentiation, as well as cell motility. Signaling by a constitutively activated Rac1 mutant deficient in PAK binding (Rac1Q61L-40C) was examined to determine whether Nischarin impinges on these other Rac1 effector pathways. Nischarin formed immunoprecipitatable complexes with Rac1Q61L and Rac1Q61L-40C when the proteins were co-expressed. In NIH3T3 cells, Rac1Q61L and Rac1Q61L-40C stimulation of a minimal NF-kappaB response element or the cyclin D1 promoter, a downstream target of NF-kappaB, was inhibited by co-expression of Nischarin. Additionally, suppression of endogenous Nischarin protein with small interfering RNA in PC12 cells enhanced Rac1Q61L and Rac1Q61L-40C activation of NF-kappaB. In further support of Nischarin suppressing PAK independent Rac signaling, foci formation in monolayers of NIH3T3 cells by Rac1Q61L-40C in cooperation with c-Raf/CAAX was inhibited by the presence of Nischarin. Nischarin alters the cellular localization of Rac1Q61L and Rac1Q61L-40C to vesicles and this positively correlates with the repression of the Rac1 signal. Thus, Nischarin, in addition to regulating the PAK strand of Rac1 signaling, can also regulate other links in the web of Rac1 signaling pathways.

Epigenetic manipulation of gene expression: a toolkit for cell biologists.

Cell biologists have been afforded extraordinary new opportunities for experimentation by the emergence of powerful technologies that allow the selective manipulation of gene expression. Currently, RNA interference is very much in the limelight; however, significant progress has also been made with two other approaches. Thus, antisense oligonucleotide technology is undergoing a resurgence as a result of improvements in the chemistry of these molecules, whereas designed transcription factors offer a powerful and increasingly convenient strategy for either up- or down-regulation of targeted genes. This mini-review will highlight some of the key features of these three approaches to gene regulation, as well as provide pragmatic guidance concerning their use in cell biological experimentation based on our direct experience with each of these technologies. The approaches discussed here are being intensely pursued in terms of possible therapeutic applications. However, we will restrict our comments primarily to the cell culture situation, only briefly alluding to fundamental differences between utilization in animals versus cells.

Raf-1 serine 338 phosphorylation plays a key role in adhesion-dependent activation of extracellular signal-regulated kinase by epidermal growth factor.

Activation of the extracellular signal-regulated kinase (ERK) 1/2 cascade by polypeptide growth factors is tightly coupled to adhesion to extracellular matrix in nontransformed cells. Raf-1, the initial kinase in this cascade, is intricately regulated by phosphorylation, localization, and molecular interactions. We investigated the complex interactions between Raf-1, protein kinase A (PKA), and p21-activated kinase (PAK) to determine their roles in the adhesion dependence of signaling from epidermal growth factor (EGF) to ERK. We conclude that Raf-1 phosphorylation on serine 338 (S338) is a critical step that is inhibited in suspended cells. Restoration of phosphorylation at S338, either by expression of highly active PAK or by expression of an S338 phospho-mimetic Raf-1 mutation, led to a partial rescue of ERK activation in suspended cells. Raf-1 inhibition in suspension was not due to excessive negative regulation on inhibitory sites S43 and S259, as these serines were largely dephosphorylated in suspended cells. Finally, strong phosphorylation of Raf-1 S338 provided resistance to PKA-mediated inhibition of ERK activation. Phosphorylation at Raf-1 S43 and S259 by PKA only weakly inhibited EGF activation of Raf-1 and ERK when cells maintained high Raf-1 S338 phosphorylation.

The integrin-binding protein Nischarin regulates cell migration by inhibiting PAK.

Nischarin, a novel intracellular protein, was originally identified as a binding partner for the alpha5beta1 integrin. Here we show that Nischarin also interacts with members of the PAK family of kinases. The amino terminus of Nischarin preferentially binds to the carboxy-terminal domain of PAK1 when the kinase is in its activated conformation. Nischarin binding to PAK1 is enhanced by active Rac, with the three proteins forming a complex, while expression of the alpha5beta1 integrin also increases the Nischarin/PAK1 association. Interaction with Nischarin strongly inhibits the ability of PAK1 to phosphorylate substrates. This effect on PAK kinase activity closely parallels Nischarin's ability to inhibit cell migration. Conversely, reduction of endogenous levels of Nischarin by RNA interference promotes cell migration. In addition, PAK1 and Nischarin colocalize in membrane ruffles, structures known to be involved in cell motility. Thus, Nischarin may regulate cell migration by forming inhibitory complexes with PAK family kinases.

Selective gene regulation with designed transcription factors: implications for therapy.

The prospect of selectively regulating gene expression is highly appealing, both for laboratory investigations and for potential therapeutic applications. This possibility has recently become a reality through the use of designed transcription factors (DTFs). This approach makes use of the DNA-binding domains of native transcription factors as scaffolds, but then introduces new DNA recognition capabilities that allow selective recognition of the regulatory regions of individual genes. The DTF strategy allows either upregulation or downregulation of transcription. This differs from approaches such as antisense or RNA interference that are intended only to inhibit gene expression. Much of the work to date on DTFs has exploited the modular nature of Cys2-His2 zinc finger (Zif) modules that, when coupled with the use of phage display or other combinatorial library techniques, allow the creation of novel and selective DNA recognition domains. These domains can then be linked to transcriptional regulatory moieties, including transactivator or repressor domains, to modulate gene expression. Recent progress has demonstrated that DTFs can selectively regulate the transcription of endogenous genes in mammalian cells and in living animals.

Plasminogen activator inhibitor-1 and -3 increase cell adhesion and motility of MDA-MB-435 breast cancer cells.

Plasminogen activator inhibitor-1 (PAI-1), an inhibitor of urokinase plasminogen activator, is paradoxically associated with a poor prognosis in breast cancer. PAI-1 is linked to several processes in the metastatic cascade. However, the role of PAI-1 in metastatic processes, which may be independent of protease inhibitory activity, is not fully understood. We report herein that PAI-1, when added exogenously to or stably transfected in human MDA-MB-435 breast carcinoma cells, had disparate effects on adhesion to extracellular matrix proteins and motility in vitro. Specifically, exogenously added PAI-1 inhibited cell adhesion to vitronectin but not fibronectin, in agreement with the literature. By contrast, stably transfected PAI-1 stimulated adhesion to both proteins. Wild-type PAI-1 was required for this stimulation, because expression of a non-protease inhibitory P14 (T333R) PAI-1 mutant failed to enhance adhesion. Compared with non-inhibitory PAI-1, wild-type PAI-1 also increased cell motility in chemotaxic assays. Furthermore, stable transfection of a related serine protease inhibitor, plasminogen activator inhibitor-3 (PAI-3, or protein C inhibitor) gave results similar to wild-type PAI-1. The stimulatory activity of PAI-3 was not seen with a non-protease inhibitory P14 PAI-3 mutant (T341R). We show that a downstream effect of endogenous wild-type PAI-1 and PAI-3 overexpression, but not their non-inhibitory counterparts, was the altered expression of alpha(2), alpha(3), alpha(4), alpha(5), and beta(1) integrin subunits. Additionally, blocking antibodies to beta(1) integrin inhibited PAI-1-induced adhesion. Our data provide experimental support for the stimulatory and inhibitory effects of PAI-1 in metastasis and introduce PAI-3 as another serpin potentially important in malignant disease.