2.4. Ras-MAPK signaling pathway

Ras proteins belong to the superfamily of monomeric GTPases, which are involved in receptor-mediated signal transduction pathways (Fig, 2). Other members of the family are Rho GTPase, a remodeller of the actin cytoskeleton, and Rab GTPase, a regulator of intracellular trafficking. Ras proteins are located on the inner surface of the plasma membrane and attached to the membrane by a farnesyl residue. Ras proteins transmit extracellular signals that promote the growth, proliferation, differentiation and survival of cells. The signaling cascade starts from the plasma membrane where the growth factor (e.g. epidermal growth factor (EGF), FGF) binds to its enzyme-linked receptor causing receptor dimerization. This leads to phosphorylation of the intracellular parts of the receptors, which activates guanine exchange factors (GEF), such as sos. GEFs are attached to the receptor by the adaptor proteins shc and grb-2. GEFs promote the exchange of GDP attached to inactive Ras to GTP, which leads to activated Ras. Ras-GTP is constantly inactivated by Ras-GAP proteins (e.g., NF1 protein, p120GAP), which promote the intrinsic GTPase of Ras. This leads to the hydrolyzation of active Ras-GTP into inactive Ras-GDP (Alberts 2002). The major downstream target of Ras-GTP is mitogen-activated protein kinase (MAPK), but it is also known to activate other targets, e.g. phosphatidylinositol 3-kinase (PI3-kinase), Ras-related guanine nucleotide dissociation stimulator (RalGDS) and phospholipase Cε (PLCε) (Rodriguez-Viciana et al. 1994, Spaargaren & Bischoff 1994, Kelley et al. 2001). Activation of MAPK occurs through specific phosphorylation of both a threonine and a tyrosine separated by a single amino acid. The first component of MAPK is called Raf, which is activated on the plasma membrane by Ras-GTP. Raf phosphorylates mitogen-activated kinase 1/2 (MEK1/2 kinase), which activates the extracellular regulated kinase 1/2 (ERK1/2 kinase or p44/42 MAPK) by phosphorylation. ERK1/2 kinase phosphorylates a variety of downstream targets, which results in changes in gene expression and the catalytic activities of enzymes (Alberts 2002). There are two other signaling pathways related to MAPK. These are called p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and they are mainly involved in transmitting inflammatory signals.

Of the members of the Ras-MAPK signaling pathway, Ras and Raf have been identified as proto-oncogenes. Gain-of-function mutations (activating mutations) of these genes drive a cell towards cancer. Proto-oncogenes with activating mutations are called oncogenes. Oncogenic Ras has been identified in every fourth cancer. The most common cancers with oncogenic Ras are pancreatic (90%), thyroid (60%) and colorectal (45%) cancers (Bos 1989). Furthermore, 35% of cancers show increased MAPK activity (Hoshino et al. 1999).

The Ras-MAPK-signaling pathway has been linked to be responsible for the malignant phenotype, including increased proliferation, defects in apoptosis, invasiveness and ability to induce neovascularization. Consequently, different therapies towards inhibiting the pathway are under development. The receptors responsible for Ras activation have proven to be effective targets for treating human cancers and are now used in clinical practice in treatment of breast cancer (Slamon et al. 2001). Therapies inhibiting Ras activation itself have also aroused a lot of interest. Farnesyl transferase inhibitors (FTI) block the farnesylation of Ras proteins, which is needed for membrane attachment and activation of Ras (Sebti & Hamilton 2000). However, these drugs have not been effective in human cancer treatment, even though they were promising in animal models (Kohl et al. 1995, End et al. 2001, Rose et al. 2001). The major downstream target of Ras, MAPK, has also been taken as a subject for drug design. MAPK inhibitors have been shown to be effective in cancer therapy in mouse models (Sebolt-Leopold et al. 1999). Inhibition of the Ras-MAPK-signaling pathway has been suggested as potential therapy for NF1. In mouse models, FTIs have been shown to reverse some abnormal features seen in NF1-deficient Schwann cells (Kim et al. 1997), but not in fibroblasts (Atit et al. 1999). Furthermore, FTIs can eliminate the learning difficulties seen in NF1 heterozygous mice (Costa et al. 2002). FTIs have also recently been tested in NF1 patients (Weiss et al. 1999).