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MAP kinase signalling pathways in cancer

A S Dhillon1, S Hagan1, O Rath1 and W Kolch1,2Top of pageAbstractCancer can be perceived as a disease of communication between and within cells. The aberrations are pleiotropic, but mitogen activated protein kinase (MAPK) pathways feature prominently. Here, we discuss recent findings and hypotheses on the role of MAPK pathways in cancer. Cancerous mutations in MAPK pathways are frequently mostly affecting Ras and B Raf in the extracellular signal regulated kinase pathway. Stress activated pathways, such as Jun N terminal kinase and p38, largely seem to counteract malignant transformation. MAPK pathways are comprised of a three tier kinase module in which a MAPK is activated upon phosphorylation by a mitogen activated protein kinase kinase (MAPKK), which in turn is activated when phosphorylated by a MAPKKK (Figure 1). To date six distinct groups of MAPKs have been characterized in mammals; extracellular signal regulated kinase (ERK)1/2, ERK3/4, ERK5, ERK7/8, Jun N terminal kinase (JNK)1/2/3 and the p38 isoforms //(ERK6)/ (Schaeffer and Weber, 1999; Chen et al., 2001b; Kyriakis and Avruch, 2001; Krens et al., 2006). The current consensus is that tumorigenesis requires deregulation of at least six cellular processes (Johnson et al., 1996), and that cancer cells have to acquire the following capabilities: independence of proliferation signals, evasion of apoptosis, insensitivity to anti growth signals, unlimited replicative potential, the ability to invade and metastasize and to attract and sustain angiogenesis for nutrient supply (Hanahan and Weinberg, 2000). To this we may add acquisition of drug resistance and avoidance of oncogene induced senescence. Abnormalities in MAPK signalling impinge on most, if not all these processes, and play a critical role in the development and progression of cancer. As the literature on MAPK pathways and cancer is huge and includes comprehensive recent reviews (Downward, 2003; Wellbrock et al., 2004; Kolch, 2005; Bradham and McClay, 2006; Galabova Kovacs et al., 2006; Kohno and Pouyssegur, 2006; Torii et al., 2006), we will take the liberty of a more subjective view and discuss emerging areas and interesting questions in the field. See text for details.

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Top of pageThe ERK pathwayThe ERK pathway is the best studied of the mammalian MAPK pathways, and is deregulated in approximately, one third of all human cancers. Historically, ERK signalling was synonymous with cell proliferation but it is now clear that that deregulation of this pathway is linked to many other aspects of the tumour phenotype. In the ERK MAPK module, ERK (ERK1 and ERK2) is activated upon phosphorylation by MEK (MEK1 and MEK2), which is itself activated when phosphorylated by Raf (Raf 1, B Raf and A Raf). ERK signalling is activated by numerous extracellular signals. The pathway whereby growth factors and mitogens activate ERK signalling is of particular relevance to cancer. In this pathway, ligand mediated activation of receptor tyrosine kinases triggers guanosine triphosphate (GTP) loading of the Ras GTPase, which can then recruit Raf kinases to the plasma membrane for activation. Most cancer associated lesions that lead to constitutive activation of ERK signalling occur at these early steps of the pathway, namely, overexpression of receptor tyrosine kinases, activating mutations in receptor tyrosine kinases, sustained autocrine or paracrine production of activating ligands, Ras mutations and B Raf mutations (Figure 2). However, there is also amplification or deregulation of its nuclear transcription factor targets, most notably myc and AP 1. fake Van Cleef & Arpels clover jewelry cheap In addition, cancer cells may switch the repertoire of extracellular matrix receptors they express to one that favours the transmission of pro growth signals. Such growth promoting integrins can activate Ras signalling (Giancotti and Ruoslahti, 1999). Thus, the fact that deregulation of this pathway in cancer occurs at several levels underlines its importance. The high frequency of activating mutations centred around the Ras axis suggests that this is the regulatory hotspot of the pathway. Indeed mathematical modelling predicts that Ras and Raf activation are very sensitive points of regulation that can determine the overall activation profile (Orton et al., 2005). Further, the fact that Ras and B Raf mutations rarely occur in the same tumour cell can be taken as indication that Raf is a main effector pathway of Ras in human carcinogenesis. However, an alternative explanation is that Ras and B Raf mutations could be synthetic lethal, and there is some evidence for that showing that co expression of mutant B Raf with mutant N Ras induces senescence (Petti et al., 2006).

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RasRas GTPases act as molecular switches that control the activity of many signalling pathways. Activating mutations in K Ras and N Ras occur in varying frequencies in different types of cancer and have been recently reviewed (Downward, 2003; Sebolt Leopold and Herrera, 2004). These mutations, invariably found at codons 12, 13 or 61, prevent efficient GTP hydrolysis, rendering Ras in an active, GTP bound state. In this conformation, Ras oncogenes can bind and activate their effectors including Raf. Although initially thought to occur mainly at the plasma membrane, there is increasing evidence that isoform specific Ras signalling can take place at different cellular compartments and within different regions of the plasma membrane (Hancock, 2003; Hancock and Parton, 2005; Philips, 2005; Mor and Philips, 2006). Such compartmentalization and trafficking of endogenous Ras oncogenes is likely to play an important role in regulating downstream signalling processes involved in tumorigenesis and is a subject that requires further investigation. For alhambra necklace replica instance, one could envision drugs that selectively target oncogenic functions of Ras by affecting its subcellular localization. It would be interesting to analyse whether the subcellular localization of mutant Ras proteins is altered in tumours.

GTP loaded Ras also recruits other molecules that play an important role nucleating an active signalling complex that is competent in activating ERK (Kolch, 2005). These complexes include scaffolds such as KSR (Therrien et al., 1996) and SUR 8/SHOC 2 (Li et al., 2000) which modulate the activation of Raf by Ras. Although no mutations in these scaffolds have been reported in human cancer, KSR knock out mice have a reduced tumour susceptibility (Nguyen et al., 2002) pointing to a role of these proteins in cancer development.

The importance of Ras proteins in a variety of tumours suggested that they would be good therapeutic targets (Sebolt Leopold and Herrera, 2004). For Ras to function as signal transducer, it has to associate with the plasma membrane. This step requires isoprenylation (farnesylation or geranylation) near the Ras C terminus. Consequently, Ras was targeted isoprenylation inhibitors. However, in the clinic these inhibitors were largely disappointing (Beeram et al., 2003; Zhu et al., 2003). The reason is not entirely clear. In part this may be related to observations suggesting that the anti tumour effects of farnesylation inhibitors are due to effects on Rho rather than due to Ras inhibition (Du and Prendergast, 1999). Further, farnesylation inhibitors do not distinguish between normal and mutant Ras, and as normal Ras can counteract the transforming action of mutant Ras (To et al., 2006), they may remove accelerator and brakes at the same time. Thus, more recent efforts have been focussed on the disrupting signalling downstream of Ras.

Raf regulationRaf kinases are direct effectors of Ras and lie at the apex of the ERK pathway kinase module. The structures of the three Raf proteins are similar, but there are salient differences how they are activated (O'Neill and Kolch, 2004; Wellbrock et al., 2004). Though sharing several common structural characteristics, the three mammalian Raf isoforms differ considerably in their modes of regulation, tissue distributions and abilities to activate MEK (Wellbrock et al., 2004). Genetic ablation of the different Raf isoforms in mice suggests that they serve mainly non redundant roles in vivo (O'Neill and Kolch, 2004; Galabova Kovacs et al., 2006). Once bound to Ras, Raf kinases are activated by a complex sequence of events involving phosphorylation, protein and protein interactions (Dhillon and Kolch, 2002; Chong et al., 2003; Wellbrock et al., 2004). These events increase the catalytic ability of Raf both by neutralising autoinhibition and facilitating activation of the kinase domain. Raf 1 activation involves a complex series of changes in phosphorylation, which entail the dephosphorylation of an inhibitory site, S259, and the phosphorylation of the N region including a critical activating site, S338, as well as phosphorylation of the activation loop for maximal activation. These sites are conserved in A Raf, and activation seems to follow a similar pattern to Raf 1. However, B Raf has already a negative charge in the N region due to twin aspartic acids and the equivalent of Raf 1 S338 is constitutively phosphorylated. Additionally, Ras alone is sufficient to activate B Raf, whereas Raf 1 requires other factors in addition. However, it is still unclear which of the Raf isoforms is required to activate ERK, and this may be different dependent on the cellular context and the stoichiometries of Raf isoforms (Galabova Kovacs et al., 2006).

B raf mutations and raf 1/b raf heterodimersB Raf has attracted enormous interest, as the b raf gene is found mutated in 66% of malignant melanomas (Davies et al., 2002), and at a lower frequency in many other human malignancies, including colon cancer, papillary thyroid cancer and serous ovarian cancer. This discovery has firmly established the involvement of Raf kinases in cancer. The most common mutation (ca. 90%) is a V600E change in the activation loop that induces the constitutive activation of catalytic activity (Wan et al., 2004). Curiously, in melanoma this mutation is rare in unexposed or chronically sun damaged skin, but frequent in skin with intermittent sun exposure and often accompanied by amplification of the mutant allele (Maldonado et al., 2003). The importance of localization is underlined by the observation that B Raf mutations do not occur in melanomas of the uvea (Spendlove et al., 2004). Further, the frequency of B Raf mutations in melanoma is positively linked with genetic variants of the melanocortin 1 receptor (Landi et al., 2006) in melanocytes, and in colorectal carcinoma (CRC) with microsatellite instability (MSI). Although it has no bearing on the good prognosis of MSI positive tumours, it is associated with poor prognosis in microsatellite stable cancers (Samowitz et al., 2005). Further, B Raf mutations in CRC correlated with a high level of multiple promoter methylation at CpG islands, whereas K Ras mutation only showed a weak association (Nagasaka et al., 2004). These findings suggest that B Raf mutations are promoted by complex genetic interactions rather than physicochemical mechanisms. They also could indicate that B Raf mutations are lethal unless a certain genetic and biochemical microenvironment permits such cells to survive.

Studies into the mechanisms of oncogenic B Raf signalling have highlighted novel mechanisms by which, Raf kinases activate MEK ERK signalling that in part differ from the classical Ras pathway. The V600E mutation drastically elevates B Raf kinase activity and its ability to activate the ERK pathway, as do most other of the cancer associated mutations (Garnett and Marais, 2004). Curiously, a few mutations do not elevate B Raf kinase activity, yet are still able to activate copy van cleef alhambra mother of pearl necklace MEK ERK signalling (Wan et al., 2004). This puzzle gave rise to recent discoveries that B Raf heterodimerizes with Raf 1 and can signal through Raf 1 (Garnett et al., 2005; Rushworth et al., 2006). These studies showed that Raf 1/B Raf heterodimerization is part of the physiological activation mechanism and contribute an important part to activation of the ERK pathway by low activity B Raf mutants, but differ in details. Rushworth et al. (2006) showed that Raf 1/B Raf heterodimerization was stimulated by mitogens, enhanced by 14 3 3, and that in the context of the heterodimer either Raf isoform could activate the other. Direct measurements of the kinase activities of the heterodimers showed that despite its low abundance it contributed to a substantial level of ERK activity. The kinase activity of the Raf 1/B Raf heterodimer towards MEK was considerably higher than the activity of B Raf or Raf 1 on their own suggesting the intriguing possibility that the heterodimer may be the main MEK activator, whereas the non heterodimeric isoforms may work in other pathways. The Marais group found that the activation of Raf in the heterodimer is one way, that is, B Raf can activate Raf, but not vice versa, and that this type of activation of Raf 1 by B Raf mutants occurs through Raf 1 activation loop phosphorylation independently of Ras (Garnett et al., 2005). This would indicate a profound difference between the physiological activation of Raf 1 that seems to obligatory require Ras (Marais et al., 1998), and the activation of Raf 1 by B Raf mutants, by implication suggesting that a tumour specific mechanism for Raf mediated MEK activation exists. This may explain why tumour cells with B Raf mutations apparently are exquisitely sensitive to MEK inhibition whereas tumour cells with Ras mutations are rather resistant (Solit et al., 2006). Conceptually, this is surprising as it would indicate that Ras can transform cells without the need to activate MEK, contradicting the tenet that B Raf is the main effector of Ras transformation. Thus, the role of Raf 1/B Raf dimerization clearly is of high interest and relevance for carcinogenesis, and warrants further investigations in order to draw firm conclusions about the molecular mechanism. In this context it is interesting to note that normal melanocytes seem to preferentially use B Raf to activate ERK, because Raf 1 activity is suppressed by cyclic AMP dependent kinase (PKA) signalling. However, Ras mutations in melanoma cells uncouple PKA from Raf 1 regulation causing a switch from B Raf to Raf 1 signalling and ERK activation becoming dependent on Raf 1 (Dumaz et al., 2006). It would be interesting to investigate whether this switch also includes changes in Raf 1/B Raf heterodimerization and whether PKA regulates heterodimer formation.

Interestingly, B Raf mutations (and less frequently activating MEK mutations) were also discovered in cardio (CFC) syndrome (Rodriguez Viciana et al., 2006), a hereditary disease hallmarked by mental retardation, congenital heart defects and abnormalities of facial structure and skin. Although the CFC mutations activate the kinase activity of B Raf comparable to the oncogenic V600E mutation, CFC patients are not predisposed to cancer. These results and the results from Raf isoform knock out mice (Galabova Kovacs et al., 2006) raise a number of important questions pertaining to the regulation of B Raf activation and signalling, including for instance whether B Raf mutants are still regulated, and whether B Raf can signal to different downstream targets depending on cell type or tissue specific modifier proteins. The existence of suppressors of mutant B Raf is suggested by the finding that >80% of benign naevi contain oncogenic B Raf mutations without ever progressing to melanoma (Pollock et al., 2003). A candidate for such a suppressor is RKIP which was originally isolated as a physiological inhibitor of Raf 1 mediated MEK phosphorylation (Yeung et al., 1999). RKIP expression is reduced in melanoma cells with mutated B Raf, and reconstitution of its expression to physiological levels suppressed the activity of the ERK pathway to normal levels and blocked cell invasion into matrigel (Schuierer et al., 2004). There are probably multiple inhibitory mechanisms that must be circumvented including escape from senescence. High level signalling by mutated B Raf can induce senescence both in human melanocytes and in congenital naevi thus preventing the mutation to induce malignant progression (Michaloglou et al., 2005).

Raf 1 mutationsIn contrast to B Raf, mutations in Raf 1 are very rare, and no A Raf mutations were found. Four Raf 1 mutations were detected in 545 established cancer cell lines (Emuss et al., 2005), but is not entirely clear whether these are polymorphisms. Only one mutant had elevated kinase activity, but failed to transform cells. Interestingly, mutating the residue equivalent to B Raf V600E also failed to produce a transforming Raf 1 protein unless a negative charge was introduced into the N region. These data show that it takes two mutations to convert Raf 1 into a transforming protein by the same mechanism as B Raf, and this may explain why B Raf is the preferred target for mutation in cancer. However, truncation or even single point mutations can confer transforming activity onto Raf 1 (Dhillon and Kolch, 2002; Wellbrock et al., 2004) indicating that there may be other reasons for the preference of B Raf mutations in cancer. Two mutations in the Raf 1 kinase domain have been found in acute myeloid leukaemia (Zebisch et al., 2006). This disease features ERK activation in more than 50% of cases, but the frequency of Raf 1 mutations was less than 1/400. One mutation activated Raf 1 whereas the other did not, although both mutants could enhance survival and induce transformation in in vitro assays, indicating that the role of Raf 1 in cancer may not rely solely on its kinase activity, but also involve kinase independent functions. These non catalytic Raf 1 functions include the counteraction of apoptosis by suppressing the proapoptotic kinases ASK 1 (Chen et al., 2001a) and MST2 (O'Neill and Kolch, 2004), and the membrane expression of Fas (Piazzolla et al., 2005), as well as the regulation of ROK to stimulate cell migration (Ehrenreiter et al., 2005).

MEK and ERK signallingActivated Raf activates MEK1 and MEK2 by phosphorylating serines 218 and 222 in the activation loop. The three Raf isoforms differ in their abilities to activate MEK1 and MEK2; B Raf is the strongest MEK kinase followed by Raf 1. A Raf is a weak MEK activator and preferentially activates MEK1, whereas Raf 1 activates both MEK1/2 with equal efficiency (Wu et al., 1996; Marais et al., 1997). Raf 1 has two separate MEK binding sites, with phosphorylation of sites in the N region strongly enhancing MEK binding (Xiang et al., 2002). The constitutive negative charge of this region in B Raf and may explain the better binding and activation of MEK by B Raf (Emuss et al., 2005). In addition, the ability of Raf to efficiently activate MEK in cells is likely to be influenced by the presence of scaffolds such as KSR (Morrison and Davis, 2003). MEK is also phosphorylated at S298 by PAK1, an event that may facilitate its coupling to Raf (Frost et al., 1997; Coles and Shaw, 2002). In addition, an inhibitory phosphorylation site on MEK, S212 was recently reported (Gopalbhai et al., 2003).

Active ERKs phosphorylate numerous cytoplasmic and nuclear targets, including kinases, phosphatases, transcription factors and cytoskeletal proteins (Yoon and Seger, 2006). ERK signalling can, depending on the particular cell type, regulate processes such diverse as proliferation, differentiation, survival, migration, angiogenesis and chromatin remodelling (Dunn et al., 2005; Yoon and Seger, 2006). A key question is how ERK can perform these different roles with high specificity and reliability. In part at least, these properties may be linked to temporal differences in the strength and localization of ERK within the cell (Murphy and Blenis, 2006). Recent studies have shown that different expression level

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