RAS signalling in the colorectum in health and disease

Abstract

RAS proteins act as molecular switches between several homeostatic inputs and signal transduction pathways that regulate important cellular processes including cell growth, differentiation and survival. Activating mutations change the function of normal proto-oncogenic RAS proteins to oncogenic RAS proteins that trigger a wide range of downstream effectors altering expression of transcription factors that together stimulate cell proliferation and modulate apoptosis and differentiation. RAS genes are amongst the most frequently mutated genes in human cancers, in particular KRAS is mutated in 40–50% of colorectal cancers. Mutation of this gene has a significant impact on treatment management and patients’ survival, particularly in relation to anti-EGFR therapy, which is only effective in KRAS wild-type cases. Here, we discuss the regulation of KRAS signalling in the colorectum, some of the post-transcriptional and post-translational modifications that control its activity, the mutations and other DNA alterations that are found in this tumour type and the implications that they have for disease progression and current drug treatments.

INTRODUCTION: THE RAS GENE FAMILY

The RAS gene family has been the focus of intense study for more than 40 years, spearheaded by the initial observation that retroviral isolates from a tumour-bearing rat were capable of inducing sarcomas in new-born rodents (Harvey, 1964). Tumour formation by the Harvey and Kirsten rat sarcoma viruses led to the identification of the H-RAS and K-RAS genes as transforming retroviral oncogenes (reviewed in Malumbres & Barbacid, 2003). The initial observation that defined genetic elements that have the ability to transform cells, was received with much scepticism, however any doubts were soon eliminated following the introduction of recombinant DNA technology and the molecular cloning of the human oncogenes HRAS and KRAS (Goldfarb, Shimizu, Perucho, & Wigler, 1982; Pulciani et al., 1982; Shih & Weinberg, 1982). Subsequent investigations identified a third human RAS gene, the NRAS oncogene, activated in human neuroblastoma and sarcoma cell lines (Hall, Marshall, Spurr, & Weiss, 1983; Shimizu, Goldfarb, Perucho, & Wigler, 1983). These three human RAS genes encode very closely related 188 or 189 amino acid length proteins, designated HRAS, NRAS and KRAS, whereas the latter generates two isoforms KRAS 4A and KRAS 4B by alternative exon splicing (Figure 1). It is well established that there are significant differences in the cell specificity and intrinsic transforming potential between different RAS oncogenes (Maher, Baker, Manning, Dibb, & Roberts, 1995).

Figure 1. RAS family polypeptide sequence similarities. Chart comparing the amino acid (AA) sequence homologies between the 4 human RAS family proteins: KRAS 4A, KRAS 4B, HRAS and NRAS. RAS protein amino acid sequences are 100% identical for the first 85 amino acids, whereas they show 85% similarity from amino acid position 85 to position 165. The hypervariable region, from amino acid position 165 to the C-terminus (position 188 or 189) displays only 4% similarity and this reflects the differences in the plasma membrane targeting domain, which is shown in detail and ends with the CAAX box (see text for further description).

RAS proteins act as molecular switches in signalling pathways

RAS proteins function as binary molecular switches involved in intracellular signalling pathways that modulate various cellular functions including proliferation, apoptosis, differentiation, cell adhesion, cytoskeletal integrity and cell migration. It is not surprising, therefore, that abnormal regulation of the RAS signalling pathway has been reported in many pathological settings, including solid tumours (Downward, 2003) and metabolic disorders, such as obesity (Hirosumi et al., 2002), heart disease (Ravingerova, Barancik, & Strniskova, 2003; Muslin, 2008) and diabetes (Osterop et al., 1992; Hirosumi et al., 2002).

RAS proteins are G proteins (or guanosine nucleotide binding proteins) that bind either GDP or GTP, and possess a weak GTPase activity. As binary switches the RAS proteins can be in the “on” state when bound to GTP and in the “off” state when bound to GDP. The extra phosphate group of the GTP holds part of the RAS polypeptide (the two switch regions, switch I and switch II) in a loaded-spring configuration. When the protein's GTPase activity cleaves the terminal (gamma) phosphate group, converting RAS-GTP (“on”) to RAS-GDP (“off”), the switch regions change shape causing the conversion to the inactive conformation (Figure 2).

Figure 2. Functional motifs of the RAS proteins. A schematic representation of the location of functional motifs, which are very similar in the 4 RAS proteins, is shown using a linear diagram of the RAS polypeptides. This shows the positions of the GTP-binding domains, the two Switch regions (Switch I and Switch II, between the downward projecting marks), the Effector-binding domain, the Hypervariable domain and the C-terminal CAAX box.

The change from the inactive (GDP-bound) to the active (GTP-bound) RAS involves activation of upstream receptors, such as receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), serpentine and cytokine receptors. These receptors serve as an integration point for several homeostatic inputs, including growth factors, cytokines, mitogens, and hormones as well as oxidative stress or heat stress (Cobb & Goldsmith, 1995). Binding of a ligand to an RTK, such as EGF receptor, triggers oligomerization of the receptor and juxtaposition of its catalytic domains in the cytoplasm. This results in the activation of its kinase activity and transphosphorylation of tyrosine moieties, which in turn leads to binding to the adaptor protein GRB2 and the recruitment of guanine nucleotide exchange factors (GEFs), such as SOS (son of sevenless) that are capable of interacting and activating RAS proteins at the cell membrane. Subcellular localization of the GEFs is important to mediate movement of the two switch regions to release GDP from RAS. Intracellular GTP is 10 fold more abundant than GDP, so GTP enters the nucleotide binding pocket of RAS and reloads the spring. When RAS protein is in the GTP-bound active conformation it has a high affinity for RAF, PI3K, RalGDS and other signalling pathway intermediates, thus transducing signal further into the cell via these and other pathways (Figure 3). To deactivate RAS-GTP the slow and inefficient intrinsic GTPase activity of RAS is greatly enhanced by the binding of GTPase activating proteins (GAPs), such as RasGAP. An inorganic phosphate is released from the GTP and the RAS protein is now bound to GDP, its shape changes back to the inactive conformation and signal transduction ceases. Hence, RAS signalling activity is regulated by the balance between GEF and GAP activity.

Figure 3. RAS signalling pathways. Initially RAS protein is inactive in the RAS-GDP bound state. Initiation of signalling through the RAS pathways occurs at the plasma membrane. Following extracellular ligand binding to membrane receptor tyrosine kinases (RTK), such as EGF binding to EGFR, receptor conformational change gives rise to receptor dimerisation and autophosphorylation. Subsequently, src-homology 2 (SH2) domains present in the GRB2 adaptor protein bind the phosphate moieties on the activated receptor. Src-homology 3 (SH3) domains in GRB2 bind proline-rich motifs present in son of sevenless (SOS – a guanosine nucleotide exchange factor or GEF), localising SOS to the inner surface of the plasma membrane. SOS interacts with RAS proteins, catalysing the exchange of GDP for GTP, thus activating RAS to a RAS-GTP state. Normally, the intrinsic weak GTPase activity of RAS is boosted by binding GTPase Activating Protein (GAP) to convert GTP to GDP, switching the complex to the inactive RAS-GDP conformation. Whilst active, RAS can transduce signals to several pathways including RAF, RalGDS and PI3K pathways. Activated RAS phosphorylates cytosolic RAF (of which there are three coding genes: ARAF, BRAF and CRAF/RAF-1, although BRAF is the active form in the large intestine). The resulting activation of RAF in turn phosphorylates cytosolic MEK, which then phosphorylates and activates MAPK (Mitogen Activated Protein Kinase, also known as ERK), leading to the subsequent downstream pathway activation with induction and repression of distinct transcription programmes, regulating cell proliferation and other processes. Mutations that activate RAS render the RAS protein products of the mutated genes constitutively active, by inhibiting or suppressing the GTPase activity of the RAS protein, thus preventing RAS from cleaving the terminal phosphate group from GTP forcing RAS to remain in the RAS-GTP active state. Activated RAS leads to increased transduction through these signalling pathways, such as the RalGDS-RAL-RALBP1 pathway. Following its activation by RAS, RalGDS activates one of the RAL proteins which in turn activates RALBP1 that can inhibit CDC42. CDC42 is a small GTPase protein of the Rho-subfamily, involved in regulating signalling pathways that control diverse cellular functions including cell morphology, migration, endocytosis and cell cycle progression. Active RAS can signal to Phosphatidylinositol 3-kinases (PI3Ks) which are a family of enzymes involved in a variety of cellular functions including cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. PI3Ks phosphorylate the inositol ring of phosphatidylinositol leading to the production of phosphatylinositol 3,4, 5-trisphosphate (PIP3) which activates AKT leading to its phosphorylation. PhosphoAKT can in turn have multiple effects via activating NFkappaB via IKK, or inhibiting BAD, Caspase 9 and AFX to suppress apoptosis and promote cell survival.

Of the RAS family of genes, there is evidence of mutational activation in around 30% of all human carcinomas and KRAS is the most frequently affected. The KRAS oncogene is usually activated in human neoplasms by point mutations (substituting one amino acid for another) at codons 12 and 13, less often at codon 61, and very infrequently or rarely at other sites such as codons 59, 146, 19 or 20 (Naguib, Wilson, Adams, & Arends, 2011). These mutations affect the regions of the RAS polypeptide that are involved in GTP-binding and GAP-binding (Figure 2). Such mutations reduce or suppress the RAS protein's intrinsic GTPase activity, thus holding it in an active GTP-bound conformation that generates increased or constitutive signalling to downstream effectors such as those involved in the RAF-MEK-MAPK, RalGDS and PI3K-AKT pathways amongst others (Figure 3) (Barbacid, 1987; Katz & McCormick, 1997).

RAS protein is attached to the plasma membrane of the cell by prenylation. The C-terminal CAAX box of RAS is farnesylated at its C (cysteine) residue in the cytosol and the RAS protein is inserted into the membrane of the endoplasmic reticulum. The tripeptide following this cysteine is AAX (where A is an aliphatic amino acid such as valine, leucine or isoleucine and X is any amino acid), which is cleaved from the C-terminus by a specific prenyl-protein protease and the new C-terminus is methylated. RAS is transported to the plasma membrane and there, it is palmitoylated, in most cases to anchor it to the membrane. However, the KRAS 4B isoform is not palmitoylated as it has a long positively charged stretch of amino acids near to the C-terminus that interact electrostatically with the membrane.

Regulation of KRAS in colorectal epithelium, including roles of miRNAs

The activation status of KRAS is regulated at various transcriptional and post-translational levels. The interaction of KRAS with downstream effectors, its cellular localization and signalling activity are governed mainly by post-translational modifications, while its transcriptional regulation is controlled by numerous transcription factors and microRNAs (Johnson et al., 2005; Akao, Nakagawa, & Naoe, 2006).

Phosphorylation and ubiquitination are both dynamic and reversible post-translational modifications that can alter the conformation, intracellular localization and activation status of KRAS. For example, PKC-driven phosphorylation of KRAS at serine-181 results in the rapid dissociation of KRAS from the plasma membrane and its association with mitochondria, where phospho-KRAS interacts with Bcl-XL to promote apoptosis (Bivona et al., 2006). Other modifications, such as monoubiquitination of KRAS at lysine-147 affects its nucleotide binding state and its interaction with downstream effectors PI3K and RAF (Sasaki et al., 2011), providing an alternative mechanism for regulating RAS activation.

At the transcriptional level, there is experimental evidence to support the existence of a transcriptional machinery for KRAS regulation that involves MAZ and PARP-1 binding with the duplex and quadruplex conformations of the G-rich nuclease hypersensitive element (GA-element) of the KRAS promoter (Cogoi, Paramasivam, Membrino, Yokoyama, & Xodo, 2010). The formation of this complex was shown to be critical for murine KRAS transcriptional activation and opens up new avenues for therapeutic intervention to repress oncogenic KRAS.

Another level of post-transcriptional regulation for the RAS oncogene is controlled by miRNAs. Initial findings have noted the involvement of the let-7 family of miRNAs targeting RAS transcripts, leading to a significant reduction in cancer progression (Johnson et al., 2005; Akao, Nakagawa, & Naoe, 2006; Kumar et al., 2008). The introduction of high-throughput miRNA profiling has led to the identification of more miRNAs, including miR-18a^*, miR-181a and miRNA-143 (Tsang & Kwok, 2009; Gao et al., 2011; Shin et al., 2011), targeting oncogenic RAS transcripts and robustly suppressing downstream signalling. One of these miRNAs, miR-18a^* interacts only with the KRAS isoform and was shown to significantly suppress proliferation and anchorage-independent growth in human cancer cells (Tsang & Kwok, 2009).

An important consequence of activated RAS signalling in vitro and in vivo is a change in the expression of a large number of genes (Luo et al., 2007). Molecular profiling of these gene signatures has helped in the characterization of the complex signalling networks that mediate the effects of activated KRAS in oncogenic transformation and cellular senescence (Bild et al., 2006; Arena et al., 2007). The combination of these approaches with high-throughput genome-wide siRNA profiling has led to the identification of many genes such as STK33, PLK1, and TBK1 that can act in a synthetic lethal manner in cells expressing oncogenic RAS (Barbie et al., 2009; Luo et al., 2009; Scholl et al., 2009). A negative selection shRNA screen on a set of genes that has previously been identified as part of a KRAS gene expression signature (n = 89) (Sweet-Cordero et al., 2005), as well as genes (n = 35) that could act as potential transcriptional regulators downstream of KRAS activation (Lamb et al., 2003), resulted in the identification of many known KRAS-effectors e.g. BRAF and RAC1 and novel regulators of oncogenic KRAS activation, such as WT1, that are critical in KRAS-induced proliferation and senescence (Vicent et al., 2010). Further identification of such links may open up new avenues for therapeutic intervention in a wide range of human cancers and highlights the importance of combining computational analysis of expression signatures with cross-species functional genomics in elucidating the complex signalling networks that drive tumour development and progression.

RAS mutations and colorectal cancer

Colorectal cancer is the second most common malignant cause of death in the UK. Colorectal carcinogenesis involves cellular transit through a spectrum of neoplastic changes, from normal mucosa to microadenomas, to adenomas with increasing dysplasia, to carcinoma. Some of the transitions are associated with characteristic genetic changes, such as APC mutation strongly associating with the earliest stage of initiation of microadenoma formation, although other genetic or epigenetic alterations may substitute for these, suggesting that the progression of colorectal cancer is complex and is likely to involve many more genetic alterations than suggested by the early Fearon and Vogelstein models (Figure 4) (Fearon & Vogelstein, 1990; Kinzler & Vogelstein, 1996).

Figure 4. Colorectal adenoma-carcinoma progression sequence. The adenoma-carcinoma progression sequence shows the transitions from normal via the earliest stage of micro-adenoma formation (monocryptal adenoma or oligocryptal adenoma) to a small but well established adenoma with low grade dysplasia, a proportion of which may progress to form a larger adenoma with high grade dysplasia, from which a malignant clone may evolve and begin invasion into the bowel wall as a carcinoma. The most frequent genetic alterations are shown, including KRAS mutations, seen in 40–50% adenomas and carcinomas, that occur mostly during the early stages of adenoma growth and progression. APC is mutated in around 60–80% adenomas and carcinomas and occurs as a very early event, whereas p53 mutation is found in approximately 40–60% cancers appearing to occur as a late event around the time of transition to carcinoma. Many other genetic or epigenetic changes can occur during this sequence and loss of a large part of chromosome 18, containing the SMAD4 and SMAD2 genes, is among the more frequent of these.

KRAS mutations can be found at all stages of colorectal tumorigenesis, including microadenomas/dysplastic aberrant crypt foci (ACF) (Shivapurkar et al., 1997), hyperplastic polyps (Otori et al., 1997), in 40–50% adenomas and 40–50% carcinomas (Ohnishi et al., 1997). KRAS mutations have been found in adenomas with increased size and dysplasia, suggesting association with abnormal growth early in carcinogenesis. The valine mutation at codon 12 of the KRAS gene (KRAS^Val12 with an activating valine substitution for glycine at codon 12,) is associated with a poorer prognosis for colorectal cancers (found in a large meta-analysis), suggesting that in some cases it confers aggressive growth properties later in carcinogenesis (Andreyev et al., 2001).

Mouse models that allow inducible expression of mutant KRAS in the murine intestinal epithelium have been successfully developed and characterised, with demonstration of KRAS^Val12 (or KRAS ^Asp12) RNA and protein expression in the small and large intestines (Luo et al. 2007, 2009, 2011a, b). These models used a mutant KRAS^Val12 transgene with an activating valine at codon 12, permitting expression of both KRAS 4A and KRAS 4B isoforms. These experimental mouse models have provided direct evidence that mutant KRAS genes expressed in intestinal epithelium do not significantly initiate intestinal adenoma growth, but they can cooperate either with other mutant genes such as APC or defective mismatch repair, or carcinogens to promote intestinal tumour formation (Sansom et al., 2006; Haigis et al., 2008; Luo et al., 2009; Luo et al., 2010; Luo, Poulogiannis, Ye, Hamoudi, & Arends, 2011a; Luo et al., 2011b).

Other RAS abnormalities in colorectal cancers, including gain/amplification of the mutated allele or deletion of the other wild-type RAS allele

Colorectal cancer is a characteristic example of solid tumours demonstrating gross chromosomal abnormalities, some of which are associated with disease progression and poor prognosis (Brosens et al., 2010; Poulogiannis et al., 2010; Nakao et al., 2011). However, unlike KRAS mutations, which are well characterized in colorectal tumorigenesis, KRAS DNA copy number gains/amplifications have a poorly defined role in the development and progression of this tumour type. Early studies in cell lines and murine models have indicated the possible implication of wild-type or mutant RAS amplification in malignant transformation, but the prevalence of such events in primary tumour samples had not been well established until recently. KRAS is located within the chromosomal region 12p12.1 and our laboratory and others have shown that this locus is the target of DNA copy number gain (3–5 copies) and amplification (≥ 5 copies) in ≈20–25% of sporadic CRCs (Brosens et al., 2010; Poulogiannis et al., 2010).

Recent data have shown that amplification of certain oncogenes (BRAF(600E) or KRAS(13D)) can confer acquired resistance to MEK1/2 inhibitors by increasing signalling through the ERK1/2 pathway (Little et al., 2011). Interestingly, the resistance phenotype could not be reverted by treating cells with the combination of ERK1/2 and PI3K inhibitors, suggesting that up-regulation of KRAS(13D) leads to the activation of multiple KRAS effector pathways, for which suitable treatments are currently lacking. Amplification of wild-type or mutant KRAS has also been detected in 15% of gastric cell lines and 5% of primary gastric tumours, and concordant overexpression of the wild-type but not the mutant allele has been associated with higher levels of GTP-bound KRAS protein. Knock-down of the wild-type KRAS transcripts in gastric cancer cell lines with amplification of this locus was associated with inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity (Mita et al., 2009).

Although, different RAS isoforms share, at least in part, common signalling pathways, leading to similar cellular responses, there are distinct differences in the functional significance of these proteins. We have recently shown that KRAS 4A has a tumour suppressor effect on carcinogen-induced murine colonic adenoma formation (Luo et al., 2010). This observation likely accounts for the selective advantage of the altered KRAS 4A : 4B isoform ratio, with reduced KRAS 4A and/or increased KRAS 4B transcript or protein levels, that is detected in a proportion of human colorectal cancers (Plowman et al., 2006; Abubaker et al., 2009). The relative expression levels of KRAS 4B transcripts were significantly increased in the adenomas of mice with complete knockout of the KRAS 4A exon (K-ras tmDelta4A/tmDelta4A) compared with heterozygous knockout of KRAS exon 4A (K-ras tmDelta4A/+) and wild-type mice (K-ras +/+) (Luo et al., 2010), suggesting that there is a complex mechanism that is regulating the expression of the two isoforms. Interestingly, KRAS 4A but not 4B overexpression has previously been associated with a better overall survival and was shown to be an independent prognostic factor in a multivariate analysis with age, gender, stage, differentiation, and MSI status (Abubaker et al., 2009).

Activation of the KRAS oncogene has also been proposed to contribute to the genesis of large-scale chromosomal abnormalities, however a direct link between RAS mutations and chromosomal instability (CIN) is still lacking (Castagnola & Giaretti, 2005). The Schizosaccharomyces pombe Ras1 pathway effectors CDC42 and SCD1 were shown to bind to microtubule regulators e.g. MOE1, which localize to the mitotic spindle (Li, Chen, & Chang, 2000; Segal & Clarke, 2001), and through this interaction they were suggested to regulate microtubule dynamic stability. Further evidence for the involvement of RAS activation in the generation of CIN came from studies of rat thyroid follicular cells, where activation of the RAS oncogene resulted in centrosome amplification and chromosome misalignment (Saavedra et al., 2000). Similar enhanced karyotypic changes were also observed in RAS-transfected 3T3 murine fibroblasts and other rodent cells in vitro (Fukasawa & Vande Woude, 1997; Orecchia et al., 2000). An interesting working hypothesis for the acquisition of chromosomal aberrations in mutant KRAS tumours involves the induction of MYC overexpression, which follows the activation of MAPK and WNT signalling pathways (McCormick, 1999). Ectopic overexpression of RAS and MYC oncoproteins was previously reported to induce higher reactive oxygen species (ROS) production (Lee et al., 1999; Tanaka et al., 2002), which in turn, has been reported to increase DNA damage (Vafa et al., 2002) and aneuploidy (Fest et al., 2002). Subsequent studies showed that MYC can induce DNA breaks in vivo and in vitro independent of increased production of ROS (Ray et al., 2006), suggesting that any CIN phenotype observed in the presence of KRAS mutation could be redox-independent. To further support this notion, recent data show that endogenous, unlike ectopic expression of the oncogenic alleles of KRAS, BRAF and MYC results in a Nrf2-dependent reduction of intracellular ROS in vivo, thereby conferring a more reduced intracellular environment associated with the activation of these oncogenes (DeNicola et al., 2011).

Although, there has been compelling evidence from in vitro studies in support of KRAS mutation involvement in CIN, results from mutant KRAS and carcinogen-induced colorectal carcinogenesis studies in rodent models do not point towards a direct link between KRAS mutation and aggravation of CIN. Most studies have reported a high frequency of KRAS G > A transitions following carcinogen-induced formation of aberrant crypt foci and adenocarcinomas, however these mutations were not associated with the presence of aneuploidy (reviewed by (Castagnola & Giaretti, 2005). Furthermore, all tumours derived from transgenic mice expressing KRAS G12V under the Villin promoter revealed a diploid DNA profile, while no colonic tumours were developed for up to eight months in transgenic mice expressing KRAS G12V under its own promoter (Janssen et al., 2002; Guerra et al., 2003). We have recently assessed the contribution of KRAS G12V mutation in the formation of CIN in a transgenic mouse model that is based on inducible Cre/LoxP intestinal epithelial expression of KRAS G12V and treatment of the large intestine with a colon-selective carcinogen 1,2-dimethylhydrazine (DMH) (Luo et al., 2011b). This study showed that mutant KRAS can promote carcinogen-induced murine colorectal tumorigenesis, however it does not alter CIN as assessed by high-resolution array-CGH analysis.

Relevance of KRAS mutation testing in human colorectal cancer therapy using anti-EGFR antibodies and future potential of anti-RAS therapies

Several pieces of evidence point to the fact that multiple effector pathways are activated following oncogenic KRAS mutation, underlying the therapeutic challenge to treat mutant KRAS-driven tumours. Data from the Kirsten RAS mutation analysis in the colorectal cancer collaborative group (RASCAL) II study have shown that out of the 12 possible KRAS mutations affecting codons 12 and 13, only the glycine to valine mutation in codon 12 has proven to have a statistically significant impact on recurrence-free and overall survival (Andreyev et al., 2001), suggesting that KRAS mutational status alone is not a good prognostic marker. The presence of KRAS mutation though is of great clinical relevance, as it has proven to play an important role in predicting the response to EGFR-inhibitor-based therapy.

There are two main classes of EGFR inhibitors currently in clinical use: the anti-EGFR monoclonal antibodies and the small-molecule EGFR tyrosine kinase inhibitors, which are not exclusive to the EGFR pathway and can block different receptor tyrosine kinases, including VEGF (Sullivan & Kozuch, 2011). Two independent clinical trials have evaluated the therapeutic effects of the anti-EGFR monoclonal antibodies panitumumab and cetuximab to show that the efficacy of both agents is confined to patients with wild-type KRAS tumours (Amado et al., 2008; Karapetis et al., 2008). Both studies concluded that the KRAS mutational status of the tumour should be assessed prior to anti-EGFR antibody therapy. Therefore, these antibodies are only administered to patients free of KRAS mutations in codons 12 and 13, which account for more than 90% of all the mutations found in colorectal cancer. The remaining of KRAS mutations are detected mostly at codons 61 and 146 (<5% each). Recent evidence indicates that the codon 61 mutation predicted for lack of response to cetuximab, similar to the lack of response with codon 12 and 13 mutations, but this was not the case for codon 146 mutation, which did not affect cetuximab efficacy (De Roock et al., 2010).

Several reports have indicated that there is heterogeneity of KRAS mutations among the different stages of tumour progression in 5–10% of patients, hence leading to mixed responses to anti-EGFR monoclonal antibody therapy (Sullivan & Kozuch, 2011). While KRAS mutation is a well-established mechanism for the primary resistance to this therapy, more than 45% of patients with wild-type KRAS tumours also fail to respond (Linardou et al., 2008), suggesting that there are multiple mechanisms that are driving resistance to this treatment. Many components of the RAS-RAF-MEK-ERK signalling cascade including NRAS and BRAF may be mutated in colorectal cancer. Interestingly, both NRAS and BRAF mutations are found in tumours with no KRAS mutation, and they too have been associated with a lack of response to anti-EGFR antibodies. However, they only account for a very small fraction of non-KRAS mutant tumours that do not respond to anti-EGFR treatment, as NRAS and BRAF mutations are detected in 2–3% and 5–8% of colorectal tumours, respectively (reviewed by (Sullivan & Kozuch, 2011).

KRAS mutations are currently being detected by various methods, many PCR-based, including gel electrophoresis assay, sequencing, high-resolution melting temperature, real-time PCR and allele-specific discrimination assays (Krens, Baas, Gelderblom, & Guchelaar, 2010). A major limitation in most of these methods include low tumour volumes and low tumour cell to normal cell ratios of the studied specimens. Therefore, it is important to establish standardized, reliable and accurate protocols for KRAS-mutation testing and screen multiple components downstream of EGFR that are likely to confer lack of response to anti-EGFR monoclonal antibody treatment.

CONCLUSION

Activation of the RAS genes has been established as one of the most frequent mutations in colorectal cancer development and progression since the 1980s. Yet, significant contributions in this field are still being made leading the way towards developing effective therapeutic treatments for RAS-mutant and RAS-wild-type tumours. The expansion of recombinant DNA technology, the availability of mouse models as well as the technological advancements in high-throughput molecular profiling of tumour specimens have all contributed to our current knowledge of RAS biology, and have opened up new avenues of investigation.

Anti-EGFR monoclonal antibody therapy is among the standard treatments for patients with metastatic colorectal cancer, yet only a subset of patients with wild-type KRAS tumours respond to this treatment. It is therefore imperative to identify novel genetic biomarkers and additional drug targets that will help overcome any drug-resistance phenotypes and ultimately tackle the substantial burden of oncogenic KRAS-driven colorectal cancer.

ACKNOWLEDGEMENTS

GP is a Pfizer Fellow of the Life Sciences Research Foundation. MJA is supported by Cancer Research UK.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.