Modelling oncogenic Ras/Raf signalling in the mouse.

The MAPK pathway is subjected to extensive regulation by posttranslational processing enzymes, phosphatases and negative feedback loops. Disruption of this regulation leads to pathway hyperactivation that mimics activating Ras mutations as has been demonstrated in mouse models lacking the Ras GAP neurofibromin (NF1) [reviewed in 47 , 48 ]. In addition to GAPs for Ras, other proteins function to attenuate the MAPK pathway such as the dual-specificity phosphatases (DUSPs) and Sprouty (Spry) proteins. Spry proteins inhibit the MAPK pathway by binding to Raf as well as to Grb2, an adaptor protein important for the recruitment of Ras GEFs [ 49 ]. Expression of Spry-1, Spry-2, and Spry-4 was increased upon K-Ras G12D expression in the lung [ 50 ], indicating that negative feedback loops are upregulated in response to oncogenic K-Ras to compensate for augmented MAPK signalling. Furthermore, Spry-2-deficiency increased the size of K-Ras G12D -induced lung tumors [ 50 ], suggesting that negative feedback regulation has tumor suppressive activity in the context of oncogenic K-Ras. Besides Spry proteins, MAPK phosphatases represent another way of negatively regulating the MAPK pathway through dephosphorylation of ERK1 and ERK2. Tyrosine, serine/threonine as well as dual-specificity phosphatases that act on ERK proteins have been identified and knockout mice have been generated to several of these [reviewed in 51 ]. However, the effect of phosphatase depletion on oncogenic Ras and Raf signalling has not yet been determined. In addition to regulation of its downstream signalling effectors, Ras itself is regulated through post-translational modification. To increase membrane affinity and promote protein interaction, Ras is modified at the carboxy-terminal CAAX motif. First, the cysteine residue is isoprenylated by farnesyltransferase (FTase), followed by endoproteolytic release by Ras converting enzyme 1 (RCE1) of the three amino acids downstream of the isoprenylcysteine. Finally, the isoprenylcysteine residue is carboxyl methylated by isoprenylcysteine carboxyl methyltransferase (ICMT). However, in the absence of FTase activity, K-Ras and N-Ras can be isoprenylated by geranylgeranyltransferase type 1 (GGTase-I), a phenomenon providing an explanation for the disappointing performance of FTase inhibitors in clinical trials. Accordingly, genetic ablation of FNTB , the gene encoding FTase, in K-Ras G12V -expressing cells did not prevent lung tumorigenesis [ 52 ]. Surprisingly, the K-Ras G12D -induced myeloproliferative disease was accelerated upon genetic deletion of RCE1 [ 53 ], suggesting that RCE1 may have a poorly understood tumor suppressor role in cells and that RCE1 is not required for Ras oncogenesis. In contrast, the lack of either GGTase-I or ICMT ameliorated the lung tumor and myeloproliferative disease-like phenotypes in K-Ras G12D mutant mice [ 54 , 55 ]. As K-Ras is still farnesylated in the setting of GGTase-I ablation, other CAAX proteins that undergo geranylgeranylation must also be required for K-Ras G12D -mediated transformation. These findings imply that GGTase-I or ICMT may represent more suitable targets RCE1 for therapeutic intervention in Ras associated malignancies, either alone or in combination with FTase inhibitors. However, the suitability of Ras-modifying enzymes as therapeutic targets needs further investigation.

Following the identification of BRAF mutations in human malignancies, novel mouse strains modelling those mutations have been developed. Pritchard and colleagues created a B-Raf V600E mouse by employing a strategy in which mice are heterozygous mutant for B-Raf in cells expressing Cre and homozygous wildtype for B-Raf in all other cells, thus closely resembling B-Raf status in human neoplasms as well as in surrounding normal tissues [ 41 ]. B-Raf V600E activation in a variety of somatic tissues by Mx1-Cre caused death in all animals within a month due to the development of nonlymphoid leukaemia of the histiocytic type [ 41 ]. Interestingly, human leukaemia has not been associated with BRAF mutations thus far. This discrepancy may represent distinct susceptibility of haematopoietic cells to oncogenic stimuli in humans and mice. A second study using a similar mouse model reported lung adenoma formation in B-Raf V600E mice after intranasal Ad-Cre instillation [ 42 ]. Even though adenomas developed with complete penetrance, they rarely progressed to adenocarcinoma, and rather demonstrated a senescence-like growth arrest following hyperplasia [ 42 ]. Interestingly, no growth arrest was observed in Kras G12D -induced lung adenomas, which eventually progressed to malignancy [ 12 ], suggesting that activation of other Ras effector pathways may be required for tumor progression (or example see ref. 57 ). B-Raf V600E could, however, cooperate with loss of p53 or p16 INK4A /p19 Arf , known mediators of senescence, to induce adenocarcinomas [ 42 ]. These studies provided experimental evidence for murine tumor initiation by mutation of B-Raf . It is expected that oncogenic B-Raf mice will prove valuable for investigating B-Raf-mediated transformation in a variety of other cancers such as melanoma, thyroid and colon cancer. Further evidence for the oncogenic properties of B-Raf was provided by a forward genetic screen employing transposon-mediated mutagenesis. In a p19 Arf -deficient background, B-Raf was the most frequently disrupted gene, leading to acceleration of sarcoma formation [ 43 ]. In the majority of cases, the T2/Onc transposon integrated in intron 9, in the orientation that directed expression of the C-terminal B-Raf kinase domain [ 43 ]. Similarly, transgenic expression of the kinase domain of Raf-1 sufficed to induce lung adenomas [ 44 ], and additional deletion of E-cadherin led to metastatic progression [ 45 ]. Furthermore, Raf-1 lacking the N-terminal negative regulatory domain cooperated with Akt activation and p19 Arf loss to promote the formation of gliomas [ 46 ]. However, expression of truncated forms of B-Raf or Raf-1 has not been associated with sporadic human malignancies and activating mutations in CRAF have not been found in cancer thus far. Even though the preclinical potential of these models may be limited for the above reasons, they have confirmed the importance of MAPK pathway activation in tumor development.

The development of preclinical mouse models has confirmed a causal role of oncogenic K-Ras expression in a variety of tumor types. Oncogenic K-Ras expression can initiate multiple malignancies, consistent with the demonstration of K-Ras mutations in different types of human preneoplasms. The cancer-initiating cell type described by Kim et al . for pulmonary adenocarcinoma [ 24 ] should now be pursued for other K-Ras-driven cancers. The finding that N-Ras and H-Ras have weak oncogenic abilities in mice was somewhat surprising and may not reflect observations made in human malignancies. Alternatively and in contrast to K-Ras, N-Ras and H-Ras may not be initiating oncogenes and may be mutated only at later stages during tumor progression. Therefore, to distinguish between these two possibilities, the acceleration of tumor progression by N-Ras or H-Ras in combination with tumor suppressor deficiency needs to be investigated. Each Ras family member is associated with different kinds of cancer. Moreover, mutant B-Raf-harbouring cancers do not completely overlap with cancers carrying Ras mutations (e.g. mutant B-Raf has not been detected in myeloid malignancies where N-Ras is the most commonly mutated Ras gene; see Table 1 ), indicating that activation of the MAPK pathway alone is unlikely to promote cancer. In fact, it has been shown in mouse models [ 56 , 57 ] and cell culture [ 58 ] that activation of other Ras effector pathways is important for tumorigenesis. The ability of endogenously expressed, mutant Ras to activate certain effector pathways may vary between the three family members and could be influenced by factors such as subcellular localization of Ras and cell type. Additionally, the oncogenic mutations may confer different activity onto Ras and Raf, which may also depend on cell type. Understanding the difference in oncogenic activities between mutant K-Ras, N-Ras, H-Ras and B-Raf as well as the cellular context in which they are transforming should ultimately explain the tropism of these oncogenes. Finally, the development of preclinical mouse models of oncogenic Ras/Raf signalling will prove instrumental for developing novel therapeutics in a timely manner.

The MAPK pathway is essential for organismal development and has been elucidated through genetic and biochemical approaches over the past several decades. The pathway is proximally activated by growth factor binding to receptor tyrosine kinases (RTK), resulting in RTK phosphorylation and activation. Consequently, adaptor molecules localize to RTKs followed by recruitment and activation of guanine nucleotide-exchange factors (GEFs). GEFs catalyse the transition from GDP-bound, inactive Ras to GTP-bound, active Ras. Ras-GTP interacts with more than a dozen effector molecules to regulate a variety of biological processes [ 1 ]. GTPase-activating proteins (GAPs) allosterically stimulate the intrinsic GTPase activity of Ras, leading to GTP hydrolysis and Ras inactivation. To activate the MAPK signalling cascade, Ras recruits Raf to the cell membrane, where Raf is activated and subsequently forms complexes with MEK, ERK and scaffolding proteins. Raf then phosphorylates MEK, which in turn phosphorylates ERK. ERK both activates cytosolic substrates and translocates to the nucleus to stimulate diverse gene expression programs through transcription factors such as JUN and ELK1 ( Figure 1 ). The recent recognition of inhibitory feedback loops through phosphatases and binding proteins with no obvious catalytic activity has added a new dimension to our understanding of the MAPK pathway. Therefore, the MAPK “pathway” is rather a complex network that is still being elucidated. The importance of the MAPK pathway in neoplasms is evident through the discovery of mutant alleles that hyperactivate the pathway in a variety of human cancers. First, oncogenic mutations in RTKs abnormally activate Ras and its downstream substrates [ 2 ]. Second, activating Ras mutations have been detected in approximately 30% of human cancers [ 3 ]. These mutations occur in codons 12, 13, and 61 and markedly diminish GTPase activity, thereby rendering Ras locked in the GTP-bound, active state. In mammals, the Ras family consists of three genes: K-Ras , N-Ras and H-Ras . While K-Ras is the predominantly mutated family member (in ~85% of cancers harboring Ras mutations), N-Ras and H-Ras mutations are relatively uncommon (~15% and <1%, respectively) [ 4 ]. Interestingly, each Ras family member is mutated in a specific subset of human malignancies: K-Ras is commonly mutated in epithelial cancers of the pancreas, lung and colon; N-Ras mutations occur frequently in melanoma, liver and myeloid malignancies; and H-Ras mutations have been observed in bladder cancer ( Table 1 ). Third, activating mutations in B-Raf , a member of the Raf family, have been discovered with high frequency in melanoma (in a non-overlapping pattern with mutations in N-Ras ) and, to a lesser extent, in thyroid, ovarian and colon cancer [ 5 – 7 ]. Importantly, activating missense mutations do not occur in the other members of the Raf family, A-Raf and Raf-1 , which may be due to their more complex mode of activation [ 8 ]. Single amino acid substitutions are sufficient to promote the active conformation of B-Raf, thereby constitutively activating the MAPK pathway. Finally, loss of negative regulators, such as members of the Sprouty family and GAPs such as NF1, can indirectly hyperactivate the MAPK signalling cascade. It is estimated that most tumors exhibit deregulation of the MAPK pathway, making it an attractive target for therapeutic intervention. In recent years, several mouse models have been generated to establish a causal relationship between MAPK mutations found in human cancer and tumor development. These mouse models have furthered our understanding of cellular transformation by aberrant MAPK signalling and offer a powerful tool for preclinical testing of novel therapeutics. In this review, we will discuss the most recent advances of mouse cancer models that involve deregulated MAPK pathway activation.