Ras (H-, K-, N-Ras)
Evidence that viral genes were translated in virus-transformed cells was first reported in the early 1970s (Green et al. 1971). Following on from these observations, it was discovered that certain 21 kDa proteins encoded by viral genes and possessing guanine nucleotide–binding properties (Scolnick et al. 1979) were essential for the maintenance of transformation in cells infected with the Kirsten and Harvey sarcoma viruses (Shih et al. 1979). Intriguingly, sequences containing a very high degree of homology to genes encoding these transforming proteins were found in normal rat, mouse, and human genomes, suggesting a physiological role, unrelated to disease, for these proteins. Mutant versions of the oncogenes associated with the Kirsten sarcoma virus (K-ras) and the Harvey sarcoma virus (H-ras) were found in cancer cell lines of various tissue origins (Der et al. 1982). The majority of these contained point mutations that resulted in the replacement of the guanosine residue at position 12. A third Ras family member termed N-ras also containing activating point mutations was subsequently identified in leukemia and neuroblastoma cell lines (Hall et al. 1983). Indeed, it was soon realized that the Ras family of proto-oncogenes are amongst the most frequently mutated genes in human cancers. The field of Ras research is now a large and complex enterprise owing to the discovery of this protein family’s fundamental roles in cell and cancer biology, development, and disease.
Structure and Function of Ras Proteins
Regulation of Ras Activity
In addition to the action of Ras GEFs and GAPs, the activity of Ras is regulated by a variety of other mechanisms. These include posttranslational modifications such as the addition of fatty acid side chains and proteolytic processing, which determine its localization. Palmitoylation of Ras at the C-terminus facilitates its association with the cell membrane (Sefton et al. 1982) and this particular localization is essential for its function. A C-terminal CAAX motif within Ras is the target of a prenylation reaction by farnesyl transferase, which is followed by cleavage of the AAX sequence, leaving a C-terminal cytosine residue. This cytosine residue is then carboxy-methylated as an essential step for full Ras function. There are slight differences in the specific modifications to K-Ras, H-Ras, and N-Ras proteins, but the final outcome of these modifications is the secure tethering of the Ras protein to the cell membrane, which is essential for its association with both upstream and downstream signaling partners (Downward 2003).
Operating upstream, GEFs such as son of sevenless (SOS) that interact with and activate Ras proteins are themselves activated by various signals including those arising from receptor tyrosine kinases (RTK) such as the EGF receptor. However, Ras may also be activated more indirectly by signals from integrins or G protein-coupled receptors (GPCR) (Fig. 2). The function of the Ras proteins as major signaling nodes is attested to by the multiple GEFs that activate them, as well as by the even larger numbers of Ras-GEF-binding partners that regulate their function. Rather unsurprisingly, Ras proteins are therefore co-opted into and required for a plethora of cellular processes.
Ras Signaling Pathways
MAP Kinase Signaling
Activated Ras interacts directly with the RAF1 protein, a Ser-Thr kinase, and stimulates its kinase activity. Most notable of the substrates of Raf are the mitogen-activated protein kinase kinases (MAPK, also known as MEKs) which phosphorylate the extracellular signal-regulated kinases (ERKs) to regulate the transcription of target genes through the E26-transcription factor proteins (ETS) (Fig. 2). The Ras-MAP kinase effector pathway regulates cell proliferation and is required for Ras-induced cell transformation (Khosravi-Far et al. 1995). Signaling through the MAP kinase pathway is frequently enhanced in cancers by the somatic acquisition of activating mutations in the Ras proteins or their effector Raf proteins. However, activating mutations in both Ras and Raf are only rarely seen together, illustrating the importance of abnormal signaling through the MAP kinase pathway in cancer (Downward 2003; Karnoub and Weinberg 2008).
PI3 Kinase Signaling
Upon activation, Ras proteins are capable of interacting with the catalytic subunit (p110) of the class I phosphoinositide 3-kinases (PI3Ks), which results in the production of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). This second messenger molecule binds a large number of proteins including PDK1 and the Ras homology family protein Rac, thereby regulating their activity. One of these pathways downstream of PtdIns(3,4,5)P3 regulates cell survival through the serine/threonine kinase AKT/protein kinase B (AKT/PKB) (Fig. 2) (Downward 2003). Like the MAP kinase pathway, the PI3 kinase signaling pathway is also indispensable for Ras-mediated cell transformation (Rodriguez-Viciana et al. 1997).
Other Ras Effector Pathways
While Ras is capable of activating RalGDS, the GEF for the Ras-like (RalA/B) small GTPases, conflicting evidence exists for the involvement of the RalA/B effector pathway in transformation. This pathway was originally considered of minor importance to transformation, but new evidence suggests that signaling through RalA/B is sufficient for transformation of some human cell types (Hamad et al. 2002). Rac1 (Khosravi-Far et al. 1995; Samuel et al. 2011), tumor invasion and metastasis inducing protein (TIAM1, a Rac-GEF protein), the epsilon form of phosphoinositide-specific phospholipase C (PLCε), and RASSF have all been shown to be Ras effectors, and oncogenic Ras has been demonstrated to cause caspase-independent cell death via autophagy (Elgendy et al. 2011), but much still remains unknown regarding other effectors downstream of Ras.
Ras in Disease
Cancers are the disease manifestations most commonly associated with aberrant signaling through Ras. Mutations in one or more of the Ras isoforms are frequently observed in cancers, with codon 12/13 mutations making up the majority of these (Loriot et al. 2009). These forms of Ras are usually able to associate with and activate effector proteins in the absence of GTP binding and indeed mutant Ras has reduced affinity for GTP. Both the MAP kinase and PI3-kinase effector pathways are required for Ras-mediated transformation and increased signaling through both these pathways been implicated in providing cancer cells with a survival advantage.
Different isoforms of Ras are preferentially expressed in different organs, leading to isoform-specific disease manifestations resulting from aberrations in Ras activity. For instance, most pancreatic cancers exhibit K-Ras mutations, but H-Ras mutations are almost never observed in this disease. Furthermore, N-Ras mutations are common in skin cancers but K-Ras mutations are not. That this is likely the result of differences in the expression levels of Ras isoforms, which may nevertheless function similarly in different cell types, is supported by the observation that the H-Ras-coding sequence placed under the transcriptional control of the endogenous K-Ras promoter can rescue the embryonic lethality resulting from K-Ras deficiency (Potenza et al. 2005).
Ras in Development
Aberrant signaling downstream of Ras has been identified as being the cause of several developmental disorders. Collectively termed cardio-facio-cutaneous diseases, neurofibromatosis type-1 and the Noonan and Costello syndromes are characterized by inherited lesions in effectors, GAPs or GEFs of Ras (Loriot et al. 2009), or infrequently by acquired somatic mutations in Ras genes. People suffering from these syndromes generally exhibit abnormalities in the bones of the face, insufficiency in cardiac function, stunted growth, and an increased cancer risk (Schubbert et al. 2007). The fact that activating germ-line K-Ras mutations are rarely observed in genetic syndromes is strong evidence that unregulated Ras activation is highly disruptive during development and is consistent with the embryonic lethality associated with inherited activating K-Ras mutations in mice.
Therapeutic Inhibition of Ras Signaling
Antagonizing the activity of mutant Ras has always been an attractive therapeutic modality for cancer. However, given the globular and relatively featureless structure of Ras and the high-binding affinity of GTP to Ras, directly targeting the protein pharmacologically has proved extremely difficult. Many strategies to target Ras have therefore focused on disrupting the posttranslational modifications essential for the localization of Ras to the cell membrane. However, these approaches have suffered from lack of selectivity. Agents such as farnesyl transferase inhibitors (FTI) also interfere with the processing of other proteins such as RhoB. Approaches to therapeutic targeting of Ras expression using antisense oligonucleotides are currently being trialed. These approaches have run into problems associated with delivery and the lack of efficient take up into cancer cells (Downward 2003). Small molecule inhibitors of the MAP kinase pathway protein Raf, such as Sorafenib (Iyer et al. 2010; Sebolt-Leopold et al. 1999) and Vemurafenib (Halaban et al. 2010; Smalley 2010), have been more successful but only target a single Ras effector pathway. Sorafenib has been approved for the treatment of renal cell and hepatocellular carcinomas and Vemurafenib for metastatic melanoma. Vigorous research is ongoing in the development of drugs targeting PI3-kinase signaling, the main issue to be overcome being lack of isoform selectivity. However, a P110δ inhibitor, Idelalisib has been approved for certain forms of leukemia and lymphoma.
Cancers expressing mutant, hyperactivated Ras are dependent on autophagy to clear protein aggregates arising from the high-energy demands placed upon the cellular machinery. A novel approach currently gaining traction and in very early stages of development is to target autophagy in combination with MAPK or PI3-kinase pathway inhibition of Ras-driven tumors (White 2012).
The Ras genes were first discovered in viruses that possess cell transforming ability before it was realized that very similar genes exist in untransformed cells. The Ras proteins have important physiological functions in transducing signals that regulate cell proliferation and survival. They constitute signaling hubs that integrate and link growth factor signals to their appropriate effector pathways. Activating mutations in Ras genes or in components of Ras effector pathways are commonly observed in cancer, leading to increased cell proliferation and survival. Germ-line mutations of Ras effector pathways underlie a group of developmental disorders including the Noonan and Costello syndromes, collectively termed cardio-facial-cutaneous syndromes. Ras-antagonizing therapies are beginning to be used effectively to treat cancers.
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