Wnt Proteins and Their Receptors

The Wnt signalling pathway is crucial in regulating cell fate during embryogenesis, cell proliferation in adults tissues and carcinogenesis. Wnt proteins released from or presented on the surface of signalling cells act on target cells by binding to the Frizzled (Fz) low density lipoprotein (LDL) receptor-related protein (LRP) complex at the cell surface (Logan and Nusse 2004). Activation of the Wnt receptors transduces a signal to several intracellular proteins, including Dishevelled, which via its association with axin prevents glycogen synthase kinase (GSK) 30 from phosphorylating critical protein substrates. Cytoplasmic P-catenin levels are normally kept low via continuous proteasome-mediated degradation, which is controlled by a complex containing GSK/APC/Axin.

When cells receive Wnt signals, the degradation pathway is inhibited, and consequently P-catenin accumulates in the cytoplasm and nucleus. Nuclear P-catenin interacts with transcription factors such as T cell-specific transcription factor (TCF) to affect transcription. Mutations and/or signals that promote constitutive activation of the Wnt signalling pathway lead to cancer. The best-known example of a disease involving a Wnt pathway mutation that produces tumours is familial adenomatous polyposis (FAP), an autosomal, dominantly inherited disease in which patients display hundreds or thousands of polyps in the colon and rectum (Nishisho et al. 1991). Mutations were also found in several cancers, including hepatocellular carcinoma (HCC) and sporadic colon cancer, indicating the uncoupling of normal P-catenin regulation from Wnt signalling control is an important event in the genesis of many cancers (Logan and Nusse 2004).

So far RNAi has emerged as a versatile strategy to study gene function and validate therapeutic targets. To translate this technology to medical use, an immediate challenge is to determine the efficacy of siRNAs in vivo. As a first step, we have evaluated the ability ofliposomal carriers such as DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulphate) to deliver active siRNA in vivo. We have assessed the intravenous delivery and found that a substantial majority of siRNA molecules were localized around the vessels 6 h after intravenous injection via the tail vein (Sioud and S0rensen 2003). Interestingly, intravenous co-administration of an anti-green fluorescent protein (anti-GFP) siRNA and a plasmid-encoding GFP-inhibited GFP expression in various organs, such as the liver and spleen (S0rensen et al. 2003). Similarly, intraperitoneal delivery of anti-TNF-a siRNA inhibited TNF-a expression in vivo and delayed the onset of septic shock following LPS injection (S0rensen et al. 2003). Of 12 recently investigated siRNAs, a siRNA targeting the mRNA site 5/-CCAACGGCAUGGAUCUCAA-3/ exhibited the greatest protective effect (Sioud and S0rensen 2004). These early findings represent a proof of principle to apply siRNA technology to disease associated with infections and/or other diseases such as cancer. Recent in vitro studies indicated that siRNA can activate immune response. In contrast to human cells, however, we have found that chemically synthesized siRNAs can be used in mice to silence gene expression without triggering an immune response. Following tail vein injection, larger dsRNAs and LPS induced inflammatory cytokine responses but not siRNA as compared to control mice (Sioud and S0rensen 2003).

Because the Wnt pathway plays an important role in tumour cell proliferation, we investigated its involvement in neuroblastoma survival. Neurob-lastomas are the most frequently occurring solid tumours in children under

5 years of age. Unfortunately, the full clinical spectrum of neuroblastomas includes very aggressive tumours, unresponsive to multi-modality treatment. Thus, novel approaches targeting molecular defects may be beneficial to neu-roblastoma patients. Targeting Wnt-1 or its receptor Fz-2 with siRNAs suppressed growth of human neuroblastoma Xenografts in mice (R. Schafer et al., submitted). Thus, interfering with Wnt signalling pathway might be a beneficial modality in neuroblastoma.

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