- About this Journal ·
- Abstracting and Indexing ·
- Advance Access ·
- Aims and Scope ·
- Annual Issues ·
- Article Processing Charges ·
- Articles in Press ·
- Author Guidelines ·
- Bibliographic Information ·
- Citations to this Journal ·
- Contact Information ·
- Editorial Board ·
- Editorial Workflow ·
- Free eTOC Alerts ·
- Publication Ethics ·
- Reviewers Acknowledgment ·
- Submit a Manuscript ·
- Subscription Information ·
- Table of Contents
Journal of Biomedicine and Biotechnology
Volume 2010 (2010), Article ID 150960, 13 pages
Clinical Relevance of KRAS in Human Cancers
1Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital, 775 20 Olomouc, Czech Republic
2Department of Experimental Medicine and Department of Human Genetics, McGill University, Montreal, Canada H3G 1A4
Received 14 August 2009; Revised 22 February 2010; Accepted 9 March 2010
Academic Editor: Dominic Fan
Copyright © 2010 Sylwia Jančík et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The KRAS gene (Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is an oncogene that encodes a small GTPase transductor protein called KRAS. KRAS is involved in the regulation of cell division as a result of its ability to relay external signals to the cell nucleus. Activating mutations in the KRAS gene impair the ability of the KRAS protein to switch between active and inactive states, leading to cell transformation and increased resistance to chemotherapy and biological therapies targeting epidermal growth factor receptors. This review highlights some of the features of the KRAS gene and the KRAS protein and summarizes current knowledge of the mechanism of KRAS gene regulation. It also underlines the importance of activating mutations in the KRAS gene in relation to carcinogenesis and their importance as diagnostic biomarkers, providing clues regarding human cancer patients' prognosis and indicating potential therapeutic approaches.
In 1982 Chang and Der, two postdoctoral fellows working in Geoffrey Cooper’s laboratory, discovered Kristen Rat Sarcoma Virus and Murine Sarcoma Virus; retroviral oncogenes related to rodent sarcoma virus genes. The human KRAS gene is a homolog of these two oncogenes . A normal form of human c-Ras has been called KRAS or KRAS2 (Kristen Rat Sarcoma Viral oncogene homolog or alternatively Kristen Murine Sarcoma Virus2 homolog). In 1983, Der described an abnormal form of the p21 protein expressed by colon and lung carcinoma cell lines and showed that the gene encoding this protein is able to transform NIH3T3 cells . This finding was later confirmed by Parada and Weinberg , who described the transformation of NIH3T3 cells by an activated KRAS oncogene. Aberrant p21 proteins were encoded by the altered KRAS gene and their expression in carcinoma tissue was causally linked to an abnormal state of activation . Since then, it has been accepted that KRAS is one of front-line sensors that initiate the activation of an array of signalling molecules allowing the transmission of transducting signals from the cell surface to the nucleus, thus affecting cell differentiation, growth, chemotaxis, and apoptosis. A signal transduction cascade initiated by the activated form of KRAS is depicted in Figure 1. As a result of these effects, KRAS elicits changes in the cytoskeleton and consequently affects cell shape, adhesion and migration [4, 5].
In the following paragraphs, KRAS protein, gene, oncogenesis, and cancer therapy is reviewed.
2. KRAS Protein
2.1. KRAS Protein Structure, Function, and Localization
KRAS belongs to a group of small GTP-binding proteins, known as the RAS superfamily or RAS-like GTPases. More than 150 RAS-like genes have been identified in mammalian genomes . The entire RAS superfamily is characterised by the presence of a catalytic G domain, but includes members with distinct evolutionary specializations with respect to different cellular process . The RAS subfamily (RAS, RHO, RAB, ARF, RAC, and RAN) includes the most frequently studied proteins, such as Harvey-Ras (H-RAS), neuroblastoma-Ras (N-RAS), and two variants of Kristen-RAS (K-RAS)—one, known as KRAS4A, which is weakly expressed in human cells and the dominant form, known as KRAS4B, which is much more highly expressed.
The KRAS gene product, KRAS protein, contains 188 amino acid residues with a molecular mass of 21.6 kD and participates in intracellular signal transduction . As mentioned above, the KRAS protein remains inactive until it binds to GTP, as depicted in Figure 2. The switch from an inactive to an active form is regulated by intracellular signals. Once the GTP is bound to the KRAS protein, KRAS undergoes conformational changes that involve two regions of the protein, thus activating it. These two important regions are known as Switch 1 (aminoacids 30–38) and Switch 2 (aminoacids 59–67), which form an effector loop, controlling the specificity of the binding of this GTPase to its effector molecules. This conformational change in the KRAS protein affects its interactions with multiple downstream transducers—GTPase-activating proteins (GAPs)—which amplify the GTPase activity of the RAS protein 100,000-fold . The change also affects interactions with guanine-exchanging/releasing factors (GEFs/GRFs) promoting the release of GTP. The KRAS protein also has intrinsic GTPase activity, stimulated by GAPs, which acts as a timer associated with direct interactions with the effectors . Mutations found in an oncogenic form of the RAS p21 protein impair GTPase activity and make the KRAS protein unresponsive to GAP proteins. Mutated forms of p21 rapidly exchange GDP for GTP, which it prefers as a substrate, thus inducing the active state. Such aberrant forms of KRAS protein deregulate many effectors, thus affecting several important cellular pathways. Many GTP derivatives targeting RAS or RAF effectors have been developed to repair the defective GTPase activity that influences the aberrant RAS signalling . However, little is known about the specificity and transport of compounds modified by GTPs through the plasma membrane.
KRAS contains four domains. The first domain includes 85 amino acids at the N-terminus and is identical in the three forms of RAS (KRAS, NRAS, and HRAS). The second domain contains 80 amino acids, with lower sequence identity (70–80%) among the three forms of RAS protein. These regions are important for the signalling function of the KRAS protein and jointly form the G-domain (amino acids 1–165, Figure 3). The G-domain of the KRAS protein includes the GTP-binding pocket, where P-loop-phosphate binding loops (aminoacids 10–16 and 56–59) interact with the b-phosphate and c-phosphate of GTP. Residues 116–119 and 152–165 interact with the guanine base. The region between amino acids 32 and 40 (the core effector region) is essential for the interactions between the putative downstream effectors and GAPs. RAS protein also contains a hypervariable region (HVR) at the C-terminus (amino acids 165–188/189; the third domain), which guides posttranslational modification and determines plasma membrane anchoring. This region plays an important role in the regulation of the biological activity of RAS protein .
Switch regions I and II play important roles in the binding of regulators and effectors. The phosphate binding pocket-P loop permits temporary binding of GTP to the RAS protein. This is also the region of GTPase activity, which negatively regulates the RAS protein via a RAS-GTP hydrolysis reaction and binding of guanosine diphosphate.
The KRAS protein acts like a plasma membrane-localized molecular switch, regulating multiple signal transduction pathways . It is synthesized in the cytosol, where it is farnesylated by farnesyl transferase at the cysteine residue of the carboxy-terminal motif CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is any amino acid). The carboxy-terminal motif forms the last domain of the KRAS protein. The AAX amino acid motif is cleaved by proteases, whereas the C-terminal carboxyl residue of the KRAS protein is methylated. Cleavage of the AXX peptide motif and methylation occur at the cytosolic surface of the endoplasmatic reticulum and are mediated by the RAS-converting enzyme Rce1 . C-terminal farnesylation plays an important role in membrane localization. In the splice variant KRAS4A, the AXX motif undergoes additional palmitoylation by palmitoyl transferase, resulting in proper targeting of KRAS4A to the membrane. However, there is no detectable palmitoylation of the predominant splice variant KRAS4B, which probably reaches the plasma membrane via a microtubule-dependent mechanism, thus avoiding the Golgi apparatus [13, 15]. Posttranslational farnesylation and carboxymethylation are believed to be important for the oncogenicity of the RAS protein. Treatment with farnesyl transferase inhibitors has been shown to inhibit anchorage-independent growth of both KRAS-transformed mouse fibroblasts and human tumour cells containing KRAS and NRAS mutations. Signal transduction of the KRAS protein does not exclusively occur at the plasma membrane. Activation of downstream signalling pathways by KRAS can also be triggered by signals from subcellular compartments, such as the endoplasmatic reticulum and the Golgi apparatus [16, 17].
2.2. Why Is the KRAS Protein an Important Target to Study?
While wild-type KRAS usually promotes cell cycle progression, it can also induce growth arrest, apoptosis, and replicative senescence when increased to abnormal levels. This can be triggered by cellular stress, ultraviolet or ionizing irradiation, heat shock, and some cytokines. In these circumstances, triggering of growth arrest can represent a defence mechanism against inappropriate activation of RAS.
It has been demonstrated that the wild-type KRAS gene is a tumour suppressor that is frequently lost during tumour progression in many types of cancer . Once the KRAS gene mutates, it acquires oncogenic properties (Table 1) and seems to be causally involved in the development of various human cancers [19, 20]. Loss of the wild-type KRAS allele has been observed in both human and mouse tumours, indicating that absence of the normal allele may facilitate transformation by one copy of the oncogenic KRAS allele . Oncogenic mutations in the KRAS gene prevent the hydrolysis of GTP, thus permanently activating the RAS molecules . Expression of a mutated KRAS gene in fibroblasts has been shown to augment metalloproteinase 2 (MMP2) expression in the matrix and enhance the invasion of cancer cells . Overexpression of this mutated form of KRAS also inhibits glycosylation of the integrin β1-chain, resulting in altered polarisation and increased adhesiveness of colon cancer cells. In addition, expression of this oncogenic form of KRAS protein has been shown to be associated with upregulated carcinoembryonic antigen (CEA) expression and disturbance of epithelial cell polarization .
3. The KRAS gene
3.1. Polymorphism/Structure and Localization of the KRAS Gene
There are two copies of the KRAS gene in the human genome, designated KRAS1 and KRAS2. The mRNA encoded by the main KRAS2 is 5.5 kb long, and differs from transcripts of the transforming Kristen murine viral gene by only six codons . Analysis of human placental and embryonic cDNA libraries has revealed that 900 bp of the KRAS1 gene is homologous to the corresponding sequence of the Kristen Murine Sarcoma Virus2 homolog, with one intervening sequence, and 300 bp of the KRAS2 is fully homologous to the viral counterpart. The KRAS1 gene is a pseudogene derived from KRAS2 by alternative mRNA splicing. Therefore, KRAS1 should be officially named KRAS1P .
In 1983 McBride and colleagues found that the protooncogenes KRAS1 and KRAS2 are localized at human chromosomes 6 and 12, respectively . Later, KRAS1 and KRAS2 were mapped by in situ hybridization to chromosome positions 6p11-12 and 12p11.1-12.1, respectively .
Sequencing showed that the KRAS2 gene has six exons. Of these, 2, 3, and 4 are invariant coding exons. Alternative splicing of exon 4 produces two mRNA forms, known as 4A and 4B. Exon 5 can be skipped during alternative splicing, giving rise to isoforms KRASA and KRASB. The 6th exon encodes the C-terminal region in KRASB and is not translated (the 3untranslated region, 3UTR) in KRASA. KRASB is the predominant splice variant of KRAS2, and is referred to, briefly, as KRAS .
There are indications that allelic losses of chromosome region 12p commonly occur in human cancers, and a frequently deleted region is near the KRAS gene at position 12p12-13 . Further, recent studies on lung adenocarcinoma suggest there is an association between the incidence of allelic losses in the 12p12-13 region and KRAS gene mutation .
3.2. Diagnostics of KRAS Mutations in Human Cancers
Diagnostics of KRAS gene mutations in clinical setting is limited by two factors: first, in the time of testing, KRAS mutated tumour cells may be in minority, outbalanced by wild type tumour cells and wild type non-tumour cells present in the sample. Second, analytically preferable snap-frozen tumour samples are rarely available for KRAS mutation testing. Instead, formalin fixed paraffin-embedded (FFPE) tissue is used. There, integrity of DNA may be severely compromised by procedure of formalin fixation (especially by its long duration and low pH).
All the known principles of DNA polymorphism detection are applicable to KRAS mutation detection and demand a dedicated review outside of the scope of this paper. More than 60 methods described can be divided into sequencing methods [30–37], methods based on specific interaction with oligonucleotide, methods based on specific interaction with enzyme [38–40], and conformational methods [41–47]. While many specificity and/or sensitivity enhancement of methods were described as well [48–53], analytical validation, systematic comparison, and assessment of methods side by side is lacking. To authors best knowledge, only Communauté Européene (CE) marked KRAS mutation detection kits are supplied by DxS (mutations in codons 12 a 13 are tested using principle of ARMS-PCR  and Scorpion primers , Vienna-Lab (reverse dot blot assay format), TIB Molbiol (KRAS LightMix clamped hybridization probes for codon 12), and Invigene (qPCR with sequence suppressor agent StopPrimer for the unwanted excess component, applicable for first two nucleotide positions in codons 12 and 13).
3.3. Regulation of KRAS Gene Expression
KRAS expression is regulated both during the initiation of transcription by the binding of proteins to its promoter and during transcriptional elongation by microRNAs affecting KRAS mRNA stability. Both human and murine KRAS gene promoters contain a nuclease hypersensitive polypurine-polypyrimidine element (NHPPE). The G-rich strand of NHPPE located in the proximal promoter sequence is able to form an intramolecular parallel G-quadruplex, consisting of three G-tetrads and three loops, which recognizes and binds nuclear proteins that are involved in transcriptional repression of KRAS expression. Accordingly, it has been reported that sequestration of nuclear proteins that bind to NHPP by an oligonucleotide mimicking the KRAS G-quadruplex resulted in 40% inhibition of KRAS transcription, compared to controls .
The transcription of KRAS is regulated, in part, by an interaction between the promoter region and the 65 kDa ESXR1 protein and, in part, by microRNAs (miRNAs). ESXR1 is a human protein with an N-terminal homeodomain in the nucleus and a C-terminal proline-rich repeat region I in the cytoplasm. The N-terminal fragment of ESXR1 binds to the TAATGTTATTA consensus sequence in exon-1 of the KRAS gene, thus inhibiting its mRNA expression . miRNAs contain a 21-22 nucleotide long noncoding sequence that is able to regulate gene expression . In 2005 it was estimated that there are more than 500 miRNAs, which collectively regulate approximately 30% of all human genes, including the RAS gene family . Regulation of gene expression by miRNAs probably occurs as a result of imperfect hybridization of the miRNA to the complementary sequences located in the 3untranslated region (3UTR) of target messenger RNA (mRNA) species. This interaction between miRNA and mRNA both decreases mRNA stability and represses protein synthesis by preventing access to ribosomes . Interestingly, many altered miRNAs have been identified in human cancers [61–63], including some of the most thoroughly analyzed miRNAs—let-7 , lin-4 , and bentam —all of which regulate cell proliferation and differentiation. Cancer-altered miRNAs can be divided into two main groups. Members of one group, the oncomirs, are upregulated in cancer and can act like oncogenes. The second group, the anti-oncomirs, probably act as tumour suppressors by targeting oncogenes, repressing the cell cycle and division of cancer cells . For example, miRNA-let7 is an oncogene-antioncomir pair that negatively regulates RAS protein levels and decreases cell proliferation rates [68, 69]. KRAS, NRAS, and HRAS harbour multiple let-7 miRNA complementary sites (LCSs) in their 3UTRs . Zhang et al.  found that reducing the activity of let-7 in HeLa cells resulted in a 70% increase in RAS protein levels, while Takamizawa et al.  found that let-7 expression was 80% lower in 60% of lung cancer adenocarcinoma and squamous cell carcinoma lines than in normal lung tissue. Moreover, a correlation between low levels of let-7 miRNA and significantly higher RAS protein expression has been found in lung squamous cell carcinomas. These results suggest that let-7 is able to downregulate the expression of RAS in human carcinomas.
These molecular findings provide a strong rationale for developing novel therapeutic treatments aimed at decreasing KRAS protein expression in cancer cells. In many cases KRAS protein expression is dramatically increased due to mutations in the KRAS gene sequence, thus making cells refractory to current therapies, such as those involving use of epidermal growth factor receptor inhibitors .
4. KRAS in Oncogenesis
Activating KRAS gene point mutations have been detected in many types of human tumours . Such oncogenic forms of the KRAS gene are prevalent in pancreatic carcinomas (>80%), colon carcinomas (40–50%), and lung carcinomas (30–50%), but are also present in biliary tract malignancies, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid leukemia [73, 74] and breast cancer .
Mutations in the KRAS gene have important effects on the process of carcinogenesis, which depend on the cells and tissues involved . The mutations found most frequently in the KRAS gene of cancer cells are located at positions 12 and 13 in exon 1, and less frequently in codons 61, 63, 117, 119, and 146 [77, 78]. These mutations are located near to the GTP binding site. Allelic mutations result in amino acid changes, namely Gly to Asp, Ala, Arg, Ser, Val, or Cys in codon 12 and Gly to Asp in codon 13 . Somatic missense mutations at positions 12, 13, 61, and 63 enable perturbation of the intrinsic GTPase activity of the KRAS protein, resulting in reductions in GTP hydrolysis capacity. Mutations in codons 12 (Figure 4) or 13 are known to lead to conformational changes in the KRAS protein.
Mutation in codon 12 of the KRAS gene causes the encoded KRAS protein to “freeze” in its active state for a much longer duration than its nonmutated counterpart . Mutations resulting in the substitution of amino acids 116, 117, 119, and 146 reduce the nucleotide affinity of the KRAS protein, thereby affecting the rate of GDP/GTP exchange. The oncogenic forms of the RAS protein have a profound effect on the downstream effector pathways, resulting in much higher proliferation rates of cancer cells expressing such forms.
The transforming ability of the KRAS oncogene may result from overexpression of the mutant KRAS allele or from deletion of the wild-type allele . Overexpression of KRAS can also be induced by the loss of p16INK4 (CDKN2A), p19INK4 (CDKN2D), or p53 . However, studies by Zhang et al. (2001) have shown that the wild-type KRAS allele can suppress the oncogenic function of the mutated allele .
In addition, the radiosensitivity of tumour cells is altered by oncogenic RAS expression, probably as a result of the effect of the KRAS mutation on several intercommunicating pathways .
4.1. Pancreatic Cancer
The prevalence of mutations in the KRAS gene at the time of diagnosis is highest in pancreatic cancers (>80% of cases), notably pancreatic adenocarcinomas predominantly harbour KRAS forms with a guanine to thymine transversion in codon 12 . Wei and colleagues examined samples collected from 30 patients with pancreatic cancer and found that 24 of them showed mutations at codon 12 and only one at codon 13 . However, concurrent KRAS mutations frequently occur in patients with pancreatic cancer . A positive association has been found in patients with pancreatic cancer between tobacco exposure and mutations in the KRAS gene . Similar associations have also been reported for coffee drinking , and milk, butter, and alcohol consumption [88, 89]. However, no direct evidence of a causal relationship between these dietary components and mutations in the KRAS gene has been presented.
4.2. Colon Cancer
The second highest incidence (about 50% of cases) of mutations in the KRAS gene is found in colon cancers [90, 91]. The progression of colon carcinomas can be divided into at least three stages. The first stage is characterized by the development of a small, benign tubular type of adenoma or polyp with sporadically detectable KRAS mutation(s) . The second stage is more aggressive and is usually associated with patches of definitive carcinoma cells, which may grow into invasive cancers characterising the third stage. Mutations of the KRAS gene have been identified in tissues from both adenoma and carcinoma cases, but at much lower frequencies in colon adenoma tissues than in carcinoma tissues [93, 94]. The incidence of mutation in the KRAS gene has been found to be low and to occur mainly in the small adenomas of patients with familial adenomatous polyposis, who have a predisposition to colon cancer  in the KRAS gene associated with colon cancer appear most often in codons 12 (28%) and 13 (8%) of exon 1 and less frequently in codon 61 . In colorectal cancer, the main substitution (Gly to Asp) has been found to occur in codon 12. Mutation from GGT (Gly) to GTT (Val) in codon 12 has been observed more frequently in primary metastatic carcinoma, suggesting that this mutation may confer a more aggressive phenotype in colorectal carcinoma . A mutation in codon 13, resulting in the substitution of Gly with Asp, observed in colon cancer has been shown to be associated with reduced survival rates . This kind of KRAS gene mutation has also been shown to occur more frequently in unstable, than in stable, colon tumours [97, 98].
4.3. Lung Cancer
Losses of KRAS wild type alleles in both mouse and human lung adenocarcinomas and squamous carcinomas have been found in many studies, notably in 67% to 100% of chemically induced murine lung adenocarcinoma cases harbouring a mutant KRAS. In humans, KRAS mutations appear in 10–30% of lung carcinoma cases, demonstrating strong associations with a history of smoking  and poor prognosis [100, 101]. Among both current and former smokers, KRAS gene mutations have been identified in 30% of lung adenocarcinoma cases. Further, although some researchers have found sporadic KRAS mutations in non-smokers with early onset of cancer, smoking history is an important factor and is correlated with increased occurrence of mutations in the KRAS gene in lung cancer cases . Mutations in the KRAS gene in codons 12 and 13 were detected in 21% of NSCLC (non-small cell lung cancer) tumour samples examined in the TRIBUTE III trial . NSCLC patients have a tendency to accumulate activating mutations in either the EGFR or KRAS genes. However, a clinical study has shown that mutations of these two genes are, in general, mutually exclusive. EGFR mutations are more often found in patients who have no history of smoking.
4.4. Breast Cancer
Although higher KRAS mutational frequency is primarily found in cancers of the pancreas, colon and lung, possible links between KRAS hyperactivity and human breast cancer have been explored recently. Hollestelle at al. found mutations in 12.5% of cases  but the Sanger COSMIC database version 28 (http://www.sanger.ac.uk/genetics/CGP/cosmic/) records only a 5% incidence . The lower frequency of KRAS mutations in breast cancer cell lines suggests that the gene mutation may be less important in breast cancer carcinogenesis than in other forms of cancer, although mutations at a “hotspot” in the KRAS gene have been found in a small subset of breast cancers.
5. KRAS and Cancer Therapy
5.1. Tyrosine Kinase Inhibitors of the EGFR1 Gene
Many clinical trials have shown that a poorer response to chemotherapy, a shorter time-to-progression, and worse overall survival are consistently associated with specific mutations in oncogenes. KRAS is one of the most frequently mutated oncogenes in many cancers, and it is also one of the most important predictors of resistance to targeted therapy using EGFR1 tyrosine kinase inhibitors (EGFR-TKIs) .
Two of the most important EGFR-specific TKIs are gefitinib (Iressa, ZD1839) and erlotinib (Tarceva). The first indications of the predictive strength of the association between the KRAS gene and therapeutic responses to the EGFR-TKI gefitinib were originally observed in NSCLC patients with tumours bearing the wild-type form of the KRAS gene and constitutively activated EGFR1 gene, due to activating mutations in exons 18 to 21 or high copy number/amplification of the EGFR1 gene. Clinically, better responses to tyrosine kinase inhibitor treatment were observed in patients with adenocarcinomas and well-differentiated tumours, female patients, non-smokers, and people of Asiatic origin [107–109]. Clinical research data show that gefitinib monotherapy is well tolerated and active against a wide range of tumour types, including colon, head, neck, breast, prostate, and lung cancers, especially NSCLCs .
EGFR-TKIs are usually used as the second line therapy in patients after failure of chemotherapy. However, gefitinib did not pass the registration procedure in the European Union because insufficient clinical benefit was demonstrated, probably because European clinical trials did not include sufficient “good responders” (Asians, women, adenocarcinoma patients and non-smokers). Clinical data also suggest that the drug represents a new therapeutic option for NSCLC patients with brain metastases .
After the successful TRIBUTE and TALENT clinical trials, erlotinib (Tarceva) was also approved by the US FDA in 2002 as a second or third line treatment for NSCLC after failure of standard chemotherapy. However, molecular analysis revealed that patients who have activating mutations in the KRAS gene (exon 1: codons 12, 13, or 61) with or without increases in EGFR copy numbers did not derive benefit from this therapy and had about a 96% chance of disease progression .
Similarly, Eberhard et al. first observed the relationship between KRAS mutations and the outcome of erlotinib therapy in a randomized phase III clinical trial in which the drug was used, in combination with first line “gold standard” chemotherapy (carboplatin and paclitaxel), to treat advanced NSCLC patients . Patients with the KRAS mutation exhibited a shorter time to progression (three months) and a shorter overall survival (four months) when treated with a combination of erlotinib and first line chemotherapy, such as treatment with cisplatin, compared to the group with wild-type KRAS, for whom the time-to-progression was 12 months. Most NSCLC patients in the erlotinib treatment study had expressed wild-type KRAS, and their KRAS status had greater prognostic than predictive value as a biomarker . However, in colorectal cancer, mutations in the KRAS gene are important predictive (as well as prognostic) biomarkers, since the effectiveness of treatment with cetuximab and panitumumab is impaired in tumours with the activating mutation. Information regarding the status of the KRAS gene allows the selection of appropriate therapies for patients who do not display activating mutations and the selection of alternative therapies for patients with mutations. Although results pertaining to the role of KRAS in the prognosis of clinical outcome or prediction of therapeutic responses to EGFR1 tyrosine kinase inhibitors are interesting, they need to be validated in larger and prospective trials, using standardized and sensitive mutation detection techniques. If the associations are confirmed, knowledge of the mutation status of KRAS in NSCLC tumours could help physicians decide which patients should receive gefitinib and/or erlotinib.
Interestingly, KRAS gene mutations also seem to provide strong predictive indication of therapeutic responses to other classes of tyrosine kinase inhibitors, as recently demonstrated for the imatinib mesylate (Glivec). Imatinib is the standard drug for patients with chronic myeloid leukaemia (CML) and patients with gastrointestinal stromal tumours (GISTs), expressing the bcr-abl fusion protein and tyrosine kinase receptor c-kit, respectively. Further, drug resistance to imatinib is usually attributed to mutation of the imatinib-binding sites of these proteins , although amplification of the bcr-abl fusion gene or overexpression of multidrug resistance proteins may be involved in some cases . However, a recent study by Agarwal et al. demonstrated that CML patients resistant to imatinib frequently expressed mutated KRAS proteins .
5.2. Monoclonal Antibodies as EGFR1 Inhibitors
Colorectal cancer is another frequent neoplasia that is associated with activation of the EGFR1 pathway, so it is not surprising that novel and successful therapeutic strategies for this condition involve EGFR1 protein kinase inhibition. In contrast to NSCLC, two monoclonal antibodies against EGFR1, rather than small molecular inhibitors of EGFR1, are generally used for treating colorectal cancer: cetuximab (Erbitux®) and panitumumab (Vectibix®). In accordance with the effect of small molecular EGFR1 inhibitors in NSCLC, KRAS alterations play a critical role in the response of colorectal cancer patients to such therapeutic monoclonal antibodies. Indeed, KRAS mutation status is the most important predictor of resistance to cetuximab or panitumumab; both the median progression-free survival of cetuximab-treated patients and overall survival was recently found to be superior in a KRAS wild-type group than in a KRAS mutant group (31 versus 10 weeks, and 16 versus 7 months, respectively) . On September 27, 2006, the US FDA approved the completely humanized monoclonal anti-EGFR1 IgG2 antibody—panitumumab (Vectibix) for clinical use in the third line treatment of patients with metastatic colorectal carcinoma who had progressed after standard chemotherapy. KRAS Panitumumab therapy was tested in a randomized study involving 463 patients , and the results showed that the wild-type KRAS gene is essential for its therapeutic activity. Progression-free survival of patients with wild-type versus mutant KRAS gene tumours was 12 versus seven weeks, while response rates obtained in another study were 17% versus 0% . These findings strongly indicate that KRAS gene status should be routinely tested as a critically important diagnostic biomarker to determine which patients will derive therapeutic benefit from EGFR1 inhibition. Indeed, analysis of the KRAS gene status in colorectal cancer cases has become conditio sinequa non for deciding whether or not to apply cetuximab or panitumumab therapy in routine clinical practice and FDA has updated Vectibix and Erbitux labels in 2009 to include this information.
5.3. Chemotherapy and Radiation
Surprisingly, the effects of KRAS gene mutations on tumour sensitivity to cytotoxic chemotherapies and radiation have only been explored in a few studies. However, expression of a 12 Val mutated form of KRAS has been shown to increase the resistance of cancer cells to radiation therapy . Similarly, the presence of oncogenic KRAS has been found to significantly increase the sensitivity of cells to a novel class of anticancer agents, cucurbitacins, in a p53- or p21-dependent manner . In contrast, an ovarian cancer cell line TOV-21G bearing a mutant allele of KRAS is reportedly significantly more sensitive to cisplatin and radiation, but not to paclitaxel or campthotecin, than the corresponding KRAS wild type line .
However, results of clinical studies by Rosell and colleagues (1995) showed that patients with a mutation in the KRAS gene had poorer clinical responses to paclitaxel monotherapy than wild type controls, suggesting that KRAS gene status is a predictive marker of paclitaxel resistance . In a phase III retrospective study on NSCLC patients (TRIBUTE), randomly treated with carboplatin and paclitaxel with erlotinib or placebo, patients with KRAS mutant tumours showed poorer clinical outcomes when treated with erlotinib plus chemotherapy compared to chemotherapy alone . An updated clinical trial (CRYSTAL) involving 540 metastatic colorectal cancer patients demonstrated that cetuximab in combination with FOLFIRI (folic acid, fluorouracil, and irinotecan) in first line therapy is highly effective against KRAS wild type, but not mutant, tumours. However, further analysis of the data showed that neither the response nor the progression-free survival of patients treated with chemotherapy alone were significantly affected by KRAS gene status, although the overall survival of patients with KRAS mutant tumours was significantly shorter than that of patients with KRAS wild type tumours . Recently we have also shown that EGFR may represent a predictive molecular marker for poor response to preoperative chemoradiotherapy in locally advanced gastric carcinoma . Responses to chemoradiotherapy were found in 60% of EGFR-negative patients, but only 13% of EGFR-positive patients (P = .044), and pathologic complete responses were observed in 29% of patients with EGFR-negative staining, but none (of eight) EGFR-positive patients (P = .16).
The above findings regarding the role of the KRAS gene in tumour responses to cytotoxic therapies appear to conflict somewhat. The predictive and prognostic significance of oncogenic KRAS seems to have been mixed in many studies, and the contributions of variations in the gene to clinical outcome appear to differ according to tumour types and therapeutic interventions. Clearly, further studies are urgently needed to confirm and clarify the findings in large prospective biomarker-oriented clinical trials.
5.4. Angiogenesis Inhibitors
Other clinical trials have also demonstrated that activating mutations in the KRAS gene can contribute to tumour progression by affecting the expression of vascular endothelial growth factor (VEGF), which plays a critical role in tumour angiogenesis. Inhibition of KRAS expression by selected KRAS antisense oligonucleotides has been shown to be associated with significantly reduced secretion of VEGF-A165 into the medium of colorectal cancer cell cultures . In addition, in a cohort of patients with pancreatic tumours, 25/33 (76%) with KRAS mutations showed higher VEGF expression, and their median survival was shorter, than those with tumours expressing the wild-type allele . Similar findings have also been reported from a study of NSCLCs, in which higher VEGF expression was observed in 50% of tumours bearing a KRAS gene mutation . Although these studies suggest that KRAS gene status could play an important role in responses to anti-VEGF targeted antiangiogenic therapy, a recent study by Hurwitz and Saini  showed that groups of patients bearing either KRAS mutant or wild-type tumours derive therapeutic benefit from first line application of the anti-VEGF monoclonal antibody bevacizumab (Avastin). Furthermore, although both groups of patients (i.e., those with wild-type KRAS and mutated KRAS genes) benefited from adding bevacizumab to chemotherapy, both progression-free survival and survival was better for wild-type KRAS patients, both with chemotherapy alone and with chemotherapy plus bevacizumab. Bevacizumab did not increase the percentage of patients with mutated KRAS who responded to treatment.
5.5. Future Therapies
An optimal therapeutic drug should be able to specifically target the mutated KRAS gene or its product, have minimal systemic toxicity and be orally active. Unfortunately, drugs like this remain to be developed and less efficient strategies need to be used in clinical trials. However, in addition to the cancer therapies mentioned above, several therapeutic agents and strategies can directly suppress the activating mutant form of the KRAS gene, and thus improve the efficiency of chemotherapy and biological therapy.
One possible approach for inhibiting KRAS expression is to use antisense oligonucleotides or viral constructs delivering antisense sequences in order to inactivate the mutant oncogene RNA message . In addition, synthesis of mutated KRAS protein has been repressed by applying a small interfering adenovirus-mediated RNA (siRNA), and the specifically designed siRNA was shown to have prolonged anti-proliferative effects against various tumour cancer cell lines expressing mutated KRAS proteins . Another, similar strategy to target mutant KRAS mRNA is based on designing an mRNA ribozyme that specifically interacts with a mutated form of the KRAS mRNA and encodes catalytic RNA molecules that bind to the mRNA substrate by base-pair complementation, leading to translation arrest and/or degradation of the specific mRNA. The KRAS-specific ribozyme strategy has also been shown to suppress successfully the proliferation of KRAS-mutated tumour cells .
Recently an interesting novel strategy employing farnesyltransferase inhibitors (FTIs) was shown to inhibit the biochemical transactivation initiated by the mutated KRAS gene. Farnesyl transferase is an enzyme that primarily regulates zinc metabolism by the addition of a farnesyl group to the cysteine residue of a protein. At least 30 proteins (including KRAS) require posttranslational farnesylation to reach their membrane positions and function properly in cell signalling. Farnesyl inhibitors represent a novel class of biologically active anticancer drugs that inhibit cell growth. After the discovery that RAS proteins have to be farnesylated to become functionally active, several farnesyl inhibitors were developed. However, Phase II and Phase III clinical trials conducted to date have found that KRAS FTIs might not be sufficient to inhibit the mutated-overactive forms of KRAS protein. The reason for this is probably incomplete inhibition of farnesylation, because garnesylation of KRAS protein by geranyltransferase I leads to suppression of the effects of farnesyltransferase inhibitors [131, 132].
It should be noted that although KRAS inhibitory strategies have shown promise in preclinical trials and have been partially successful in clinical trials, there are insufficient data on their efficacy in combination with anti-EGFR1 strategies to recommend their routine use as yet.
The evidence from various studies summarized in this review demonstrates that the KRAS protein is an important signal transducer involved in the regulation of various cellular responses during cell proliferation, differentiation, and survival. A pivotal function of KRAS protein in the regulation of the MAPK and PI3K/AKT pathways is its effect on the proliferation rate of both normal and cancer cells. Activating mutations of the KRAS protein, which frequently occur in cancer cells, indicate poorer prognosis and increased resistance to some cancer therapies.
Overall, this review summarizes novel approaches allowing the management of cancers with or without KRAS mutations, and highlights the importance of early identification of somatic mutations in the KRAS gene in cancer biopsies. The latter assists oncologists to select the best available therapies for their cancer patients.
This work was supported by grants from the Ministry of Education, Youth, and Sports, (MSM 6198959216 and LC07017) and the Ministry of Health of the Czech Republic (NSNR9076 and NS9959). Infrastructural part of this project (Institute of Molecular and Translational Medicine) was supported from the Operational Programme Research and Development for Innovations (project CZ.1.05/2.1.00/01.0030). S. Jančík and J. Drábek both contributed equally.
- E. H. Chang, M. A. Gonda, R. W. Ellis, et al., “Human genome contains four genes homologous to transforming genes of Harvey and Kirsten murine sarcoma viruses,” Proceedings of the National Academy of Sciences of the United States of America, vol. 79, no. 16, pp. 4848–4852, 1982.
- C. J. Der and G. M. Cooper, “Altered gene products are associated with activation of cellular genes in human lung and colon carcinomas,” Cell, vol. 32, no. 1, pp. 201–208, 1983.
- L. F. Parada and R. A. Weinberg, “Presence of a Kirsten murine sarcoma virus ras oncogene in cells transformed by 3-methylcholanthrene,” Molecular & Cellular Biology, vol. 3, no. 12, pp. 2298–2301, 1983.
- D. Esser, B. Bauer, R. M. F. Wolthuis, A. Wittinghofer, R. H. Cool, and P. Bayer, “Structure determination of the ras-binding domain of the Ral-specific guanine nucleotide exchange factor Rlf,” Biochemistry, vol. 37, no. 39, pp. 13453–13462, 1998.
- J. Zuber, O. I. Tchernitsa, B. Hinzmann, et al., “A genome-wide survey of RAS transformation targets,” Nature Genetics, vol. 24, no. 2, pp. 144–152, 2000.
- K. Wennerberg, K. L. Rossman, and C. J. Der, “The Ras superfamily at a glance,” Journal of Cell Science, vol. 118, no. 5, pp. 843–846, 2005.
- M. Paduch, F. Jeleń, and J. Otlewski, “Structure of small G proteins and their regulators,” Acta Biochimica Polonica, vol. 48, no. 4, pp. 829–850, 2001.
- J. P. McGrath, D. J. Capon, and D. H. Smith, “Structure and organization of the human Ki-ras proto-oncogene and a related processed pseudogene,” Nature, vol. 304, no. 5926, pp. 501–506, 1983.
- P. Gideon, J. John, M. Frech, et al., “Mutational and kinetic analyses of the GTPase-activating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity,” Molecular and Cellular Biology, vol. 12, no. 5, pp. 2050–2056, 1992.
- C. Giglione, M. C. Parrini, S. Baouz, A. Bernardi, and A. Parmeggiani, “A new function of p120-GTPase-activating protein: prevention of the guanine nucleotide exchange factor-stimulated nucleotide exchange on the active form of Ha-Ras p21,” The Journal of Biological Chemistry, vol. 272, no. 40, pp. 25128–25134, 1997.
- K. Scheffzek, M. R. Ahmadian, W. Kabsch, et al., “The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic ras mutants,” Science, vol. 277, no. 5324, pp. 333–338, 1997.
- J. F. Hancock and I. A. Prior, “Electron microscopic imaging of Ras signaling domains,” Methods, vol. 37, no. 2, pp. 165–172, 2005.
- J. F. Hancock, A. I. Magee, J. E. Childs, and C. J. Marshall, “All ras proteins are polyisoprenylated but only some are palmitoylated,” Cell, vol. 57, no. 7, pp. 1167–1177, 1989.
- K. Rajalingam, R. Schreck, U. R. Rapp, and Š. Albert, “Ras oncogenes and their downstream targets,” Biochimica et Biophysica Acta, vol. 1773, no. 8, pp. 1177–1195, 2007.
- J. A. Thissen, J. M. Gross, K. Subramanian, T. Meyer, and P. J. Casey, “Prenylation-dependent association of Ki-Ras with microtubules. Evidence for a role in subcellular trafficking,” The Journal of Biological Chemistry, vol. 272, no. 48, pp. 30362–30370, 1997.
- S. R. Hingorani, E. F. Petricoin III, A. Maitra, et al., “Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse,” Cancer Cell, vol. 4, no. 6, pp. 437–450, 2003.
- M. Fivaz and T. Meyer, “Reversible intracellular translocation of KRAS but not HRas in hippocampal neurons regulated by C/calmodulin,” Journal of Cell Biology, vol. 170, no. 3, pp. 429–441, 2005.
- Z. Zhang, Y. Wang, H. G. Vikis, et al., “Wildtype KRAS2 can inhibit lung carcinogenesis in mice,” Nature Genetics, vol. 29, no. 1, pp. 25–33, 2001.
- M. S. McCoy, J. J. Toole, J. M. Cunningham, et al., “Characterization of a human colon/lung carcinoma oncogene,” Nature, vol. 302, no. 5903, pp. 79–81, 1983.
- O. Kranenburg, “The KRAS oncogene: past, present, and future,” Biochimica et Biophysica Acta, vol. 1756, no. 2, pp. 81–82, 2005.
- M. E. Hegi, T. R. Devereux, W. F. Dietrich, et al., “Allelotype analysis of mouse long carcinomas reveals frequent allelic losses on chromosome 4 and an association between allelic imbalances on chromosome 6 and K-ras activation,” Cancer Research, vol. 54, no. 23, pp. 6257–6264, 1994.
- M. Barbacid, “Ras genes,” Annual Review of Biochemistry, vol. 56, pp. 779–827, 1987.
- J. Liao, J. C. Wolfman, and A. Wolfman, “K-Ras regulates the steady-state expression of matrix metalloproteinase 2 in fibroblasts,” The Journal of Biological Chemistry, vol. 278, no. 34, pp. 31871–31878, 2003.
- Z. Yan, X. Deng, M. Chen, et al., “Oncogenic c-Ki-ras but not oncogenic c-Ha-ras up-regulates CEA expression and disrupts basolateral polarity in colon epithelial cells,” The Journal of Biological Chemistry, vol. 272, no. 44, pp. 27902–27907, 1997.
- O. W. McBride, D. C. Swan, S. R. Tronick, et al., “Regional chromosomal localization of N-ras, K-ras-1, K-ras-2 and myb oncogenes in human cells,” Nucleic Acids Research, vol. 11, no. 23, pp. 8221–8236, 1983.
- N. C. Popescu, S. C. Amsbaugh, and J. A. DiPaolo, “Chromosomal localization of three human ras genes by in situ molecular hybridization,” Somatic Cell and Molecular Genetics, vol. 11, no. 2, pp. 149–155, 1985.
- C. Carta, F. Pantaleoni, G. Bocchinfuso, et al., “Germline missense mutations affecting KRAS isoform B are associated with a severe Noonan syndrome phenotype,” American Journal of Human Genetics, vol. 79, no. 1, pp. 129–135, 2006.
- S. Takeuchi, C. R. Bartram, C. W. Miller, et al., “Acute lymphoblastic leukemia of childhood: identification of two distinct regions of deletion on the short arm of chromosome 12 in the region of TEL and KIP1,” Blood, vol. 87, no. 8, pp. 3368–3374, 1996.
- J. Li, Z. Zhang, Z. Dai, et al., “LOH of chromosome 12p correlates with KRAS2 mutation in non-small cell lung cancer,” Oncogene, vol. 22, no. 8, pp. 1243–1246, 2003.
- S. Ogino, T. Kawasaki, M. Brahmandam, et al., “Sensitive sequencing method for KRAS mutation detection by pyrosequencing,” Journal of Molecular Diagnostics, vol. 7, no. 3, pp. 413–421, 2005.
- E. Khanifar and P. M. Ward, “Modified mutagenic PCR-RFLP and PCR-ASO methods for the detection of KRAS codon 12 and 13 mutations in colorectal cancer,” Journal of Molecular Diagnostics, vol. 10, no. 6, p. 599, 2008.
- J. W. Moul, S. M. Theune, and E. H. Chang, “Detection of RAS mutations in archival testicular germ cell tumors by polymerase chain reaction and oligonucleotide hybridization,” Genes Chromosomes & Cancer, vol. 5, no. 2, pp. 109–118, 1992.
- D.-B. Zhu, D. Xing, X. Li, and L. Zhang, “Rapid detection of K-ras oncogene point mutation by real-time fluorescence allele-specific amplification,” Chemical Journal of Chinese Universities, vol. 28, no. 6, pp. 1031–1034, 2007.
- J.-H. Park, I.-J. Kim, H. C. Kang, et al., “Oligonucleotide microarray-based mutation detection of the K-ras gene in colorectal cancers with use of competitive DNA hybridization,” Clinical Chemistry, vol. 50, no. 9, pp. 1688–1691, 2004.
- M. B. Wabuyele, H. Farquar, W. Stryjewski, et al., “Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes,” Journal of the American Chemical Society, vol. 125, no. 23, pp. 6937–6945, 2003.
- C. Fernandez-Vega, D. C. Garcia-Olmo, M. A. Ballesteros, and D. Garcia-Olmo, “Development of a simple and sensitive technique for detection of point mutations in the K-ras oncogene,” Applied Biochemistry and Biotechnology Part B, vol. 22, no. 2, pp. 115–121, 2002.
- M. Albitar, W. C. Wu, E. Feltz, et al., “Simplified reverse dot blot analyses for detecting ras oncogene mutations,” Molecular Diagnosis, vol. 2, no. 3, pp. 169–176, 1997.
- S. Bentivegna, J. Zheng, E. Namsaraev, et al., “Rapid identification of somatic mutations in colorectal and breast cancer tissues using mismatch repair detection (MRD),” Human Mutation, vol. 29, no. 3, pp. 441–450, 2008.
- G. Amicarelli, E. Shehi, M. G. Makrigiorgos, and D. Adlerstein, “FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations,” Nucleic Acids Research, vol. 35, no. 19, article e131, 2007.
- G. Amicarelli, D. Adlerstein, E. Shehi, F. Wang, and G. M. Makrigiorgos, “Genotype-specific signal generation based on digestion of 3-way DNA junctions: application to KRAS variation detection,” Clinical Chemistry, vol. 52, no. 10, pp. 1855–1863, 2006.
- P. O. Ekstrom and J. Bjorheim, “Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis,” Electrophoresis, vol. 27, no. 10, pp. 1878–1885, 2006.
- J. Bjorheim, M. Minarik, G. Gaudernack, and P. O. Ekstrom, “Mutation detection in KRAS exon 1 by constant denaturant capillary electrophoresis in 96 parallel capillaries,” Analytical Biochemistry, vol. 304, no. 2, pp. 200–205, 2002.
- M. Minarik, L. Minarikova, M. Hrabikova, P. Minarikova, P. Hrabal, and M. Zavoral, “Application of cycling gradient capillary electrophoresis to detection of APC, K-ras, and DCC point mutations in patients with sporadic colorectal tumors,” Electrophoresis, vol. 25, no. 7-8, pp. 1016–1021, 2004.
- C. F. Taylor, “Mutation scanning using high-resolution melting,” Biochemical Society Transactions, vol. 37, no. 2, pp. 433–437, 2009.
- H. Zou, W. R. Taylor, J. J. Harrington, et al., “High detection rates of colorectal neoplasia by stool DNA testing with a novel digital melt curve assay,” Gastroenterology, vol. 136, no. 2, pp. 459–470, 2009.
- Y. Bian, H. Matsubayashi, C. Pin-Li, et al., “Detecting low-abundance p16 and p53 mutations in pancreatic juice using a novel assay: heteroduplex analysis of limiting dilution PCRs,” Cancer Biology and Therapy, vol. 5, no. 10, pp. 1392–1399, 2006.
- Y. Endo, L. Zhang, R. Katashima, et al., “Effect of polymer matrix and glycerol on rapid single-strand conformation polymorphism analysis by capillary and microchip electrophoresis for detection of mutations in K-ras gene,” Electrophoresis, vol. 26, no. 17, pp. 3380–3386, 2005.
- S. K. Banerjee, W. F. Makdisi, A. P. Weston, and D. R. Campbell, “A two-step enriched-nested PCR technique enhances sensitivity for detection of codon 12 K-ras mutations in pancreatic adenocarcinoma,” Pancreas, vol. 15, no. 1, pp. 16–24, 1997.
- E. H. Lim, S.-L. Zhang, J.-L. Li, et al., “Using whole genome amplification (WGA) of low-volume biopsies to assess the prognostic role of EGFR, KRAS, p53, and CMET mutations in advanced-stage non-small cell lung cancer (NSCLC),” Journal of Thoracic Oncology, vol. 4, no. 1, pp. 12–21, 2009.
- J. Li and G. M. Makrigiorgos, “COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing,” Biochemical Society Transactions, vol. 37, no. 2, pp. 427–432, 2009.
- M. Beau-Faller, M. Legrain, A.-C. Voegeli, et al., “Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping,” British Journal of Cancer, vol. 100, no. 6, pp. 985–992, 2009.
- K. Tatsumi, Y. Mitani, J. Watanabe, et al., “Rapid screening assay for KRAS mutations by the modified smart amplification process,” Journal of Molecular Diagnostics, vol. 10, no. 6, pp. 520–526, 2008.
- P. Nollau, C. Fischer, P. Tschentscher, and C. Wagener, “Enrichment of mutant alleles by chromatographic removal of wild type alleles: a new principle for the detection of alleles with unknown point mutations at excess of wild type alleles,” Clinical Chemistry and Laboratory Medicine, vol. 37, no. 9, pp. 877–881, 1999.
- C. R. Newton, A. Graham, L. E. Heptinstall, et al., “Analysis of any point mutation in DNA. The aplivication refractory mutation system (ARMS),” Nucleic Acids Research, vol. 17, no. 7, pp. 2503–2516, 1989.
- N. Thelwell, S. Millington, A. Solinas, J. Booth, and T. Brown, “Mode of action and application of Scorpion primers to mutataon detection,” Nucleic Acids Research, vol. 28, no. 19, pp. 3752–3761, 2000.
- S. Cogoi and L. E. Xodo, “G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription,” Nucleic Acids Research, vol. 34, no. 9, pp. 2536–2549, 2006.
- M. Yanagihara, S. Ishikawa, M. Naito, J. Nakajima, H. Aburatani, and M. Hatakeyama, “Paired-like homeoprotein ESXR1 acts as a sequence-specific transcriptional repressor of the human K-ras gene,” Oncogene, vol. 24, no. 38, pp. 5878–5887, 2005.
- M. Lagos-Quintana, R. Rauhut, W. Lendeckel, and T. Tuschl, “Identification of novel genes coding for small expressed RNAs,” Science, vol. 294, no. 5543, pp. 853–858, 2001.
- B. P. Lewis, C. B. Burge, and D. P. Bartel, “Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets,” Cell, vol. 120, no. 1, pp. 15–20, 2005.
- E. C. Lai, “Micro RNAs are complementary to UTR sequence motifs that mediate negative post-transcriptional regulation,” Nature Genetics, vol. 30, no. 4, pp. 363–364, 2002.
- M. Z. Michael, S. M. O'Connor, N. G. Van Holst Pellekaan, G. P. Young, and R. J. James, “Reduced accumulation of specific microRNAs in colorectal neoplasia,” Molecular Cancer Research, vol. 1, no. 12, pp. 882–891, 2003.
- W. Tam, S. H. Hughes, W. S. Hayward, and P. Besmer, “Avian bic, a gene isolated from a common retroviral site in avian leukosis virus-induced lymphomas that encodes a noncoding RNA, cooperates with c-myc in lymphomagenesis and erythroleukemogenesis,” Journal of Virology, vol. 76, no. 9, pp. 4275–4286, 2002.
- G. A. Calin, C. Sevignani, C. D. Dumitru, et al., “Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 9, pp. 2999–3004, 2004.
- B. J. Reinhart, F. J. Slack, M. Basson, et al., “The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans,” Nature, vol. 403, no. 6772, pp. 901–906, 2000.
- R. C. Lee, R. L. Feinbaum, and V. Ambros, “The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14,” Cell, vol. 75, no. 5, pp. 843–854, 1993.
- J. Brennecke, D. R. Hipfner, A. Stark, R. B. Russell, and S. M. Cohen, “bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila,” Cell, vol. 113, no. 1, pp. 25–36, 2003.
- L. P. Lim, N. C. Lau, P. Garrett-Engele, et al., “Microarray analysis shows that some microRNAs downregulate large numbers of-target mRNAs,” Nature, vol. 433, no. 7027, pp. 769–773, 2005.
- J. Takamizawa, H. Konishi, K. Yanagisawa, et al., “Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival,” Cancer Research, vol. 64, no. 11, pp. 3753–3756, 2004.
- A. Goga and C. Benz, “Anti-oncomir suppression of tumor phenotypes,” Molecular Interventions, vol. 7, no. 4, pp. 199–202, 2007.
- S. M. Johnson, H. Grosshans, J. Shingara, et al., “RAS is regulated by the let-7 microRNA family,” Cell, vol. 120, no. 5, pp. 635–647, 2005.
- E. Massarelli, M. Varella-Garcia, X. Tang, et al., “KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer,” Clinical Cancer Research, vol. 13, no. 10, pp. 2890–2896, 2007.
- M. W. Anderson, S. H. Reynolds, M. You, and R. M. Maronpot, “Role of Proto-oncogene activation in carcinogenesis,” Environmental Health Perspectives, vol. 98, pp. 13–24, 1992.
- J. L. Bos, E. R. Fearon, and S. R. Hamilton, “Prevalence of ras gene mutations in human colorectal cancers,” Nature, vol. 327, no. 6120, pp. 293–297, 1987.
- J. Downward, “Role of receptor tyrosine kinases in G-protein-coupled receptor regulation of Ras: transactivation or parallel pathways?” Biochemical Journal, vol. 376, pp. e9–e10, 2003.
- S. Schubbert, K. Shannon, and G. Bollag, “Hyperactive Ras in developmental disorders and cancer,” Nature Reviews Cancer, vol. 7, no. 4, pp. 295–308, 2007.
- C. Guerra, N. Mijimolle, A. Dhawahir, et al., “Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context,” Cancer Cell, vol. 4, no. 2, pp. 111–120, 2003.
- M. Barbacid, “ras oncogenes: their role in neoplasia,” European Journal of Clinical Investigation, vol. 20, no. 3, pp. 225–235, 1990.
- S. Rodenhuis and R. J. C. Slebos, “The ras oncogenes in human lung cancer,” American Review of Respiratory Disease, vol. 142, no. 6, part 2, pp. S27–S30, 1990.
- S. J. Clayton, F. M. Scott, J. Walker, et al., “K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification,” Clinical Chemistry, vol. 46, no. 12, pp. 1929–1938, 2000.
- C. J. Marshall, “How does p transform cells?” Trends in Genetics, vol. 7, no. 3, pp. 91–95, 1991.
- M. Serrano, “The tumor suppressor protein p,” Experimental Cell Research, vol. 237, no. 1, pp. 7–13, 1997.
- R. Rengan, K. A. Cengel, and S. M. Hahn, “Clinical target promiscuity: lessons from ras molecular trials,” Cancer and Metastasis Reviews, vol. 27, no. 3, pp. 403–414, 2008.
- D. Li, P. F. Firozi, W. Zhang, et al., “DNA adducts, genetic polymorphisms, and K-ras mutation in human pancreatic cancer,” Mutation Research, vol. 513, no. 1-2, pp. 37–48, 2002.
- S. Wei, Z. Liang, J. Gao, et al., “Patterns of K-ras codon 12 and 13 mutations found in pancreatic adenocarcinoma of 30 Chinese patients by microdissection, PCR and direct sequencing,” Journal of Gastroenterology and Hepatology, vol. 20, no. 1, pp. 67–72, 2005.
- L. Laghi, O. Orbetegli, P. Bianchi, et al., “Common occurrence of multiple K-RAS mutations in pancreatic cancers with associated precursor lesions and in biliary cancers,” Oncogene, vol. 21, no. 27, pp. 4301–4306, 2002.
- R. H. Hruban, A. D. M. van Mansfeld, G. J. A. Offerhaus, et al., “K-ras oncogene activation in adenocarcinoma of the human pancreas: a study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization,” American Journal of Pathology, vol. 143, no. 2, pp. 545–554, 1993.
- M. Porta, N. Malats, L. Guarner, et al., “Association between coffee drinking and K-ras mutations in exocrine pancreatic cancer,” Journal of Epidemiology and Community Health, vol. 53, no. 11, pp. 702–709, 1999.
- N. Malats, M. Porta, J. M. Corominas, et al., “Ki-ras mutations in exocrine pancreatic cancer: association with clinico-pathological characteristics and with tobacco and alcohol consumption,” International Journal of Cancer, vol. 70, no. 6, pp. 661–667, 1997.
- E. Morales, M. Porta, J. Vioque, et al., “Food and nutrient intakes and K-ras mutations in exocrine pancreatic cancer,” Journal of Epidemiology and Community Health, vol. 61, no. 7, pp. 641–649, 2007.
- V. Bazan, V. Agnese, S. Corsale, et al., “Specific TP53 and/or Ki-ras mutations as independent predictors of clinical outcome in sporadic colorectal adenocarcinomas: results of a 5-year Gruppo Oncologico dell'Italia Meridionale (GOIM) prospective study,” Annals of Oncology, vol. 16, supplement 4, pp. iv50–iv55, 2005.
- J. Benhattar and E. Saraga, “Molecular genetics of dysplasia in ulcerative colitis,” European Journal of Cancer Part A, vol. 31, no. 7-8, pp. 1171–1173, 1995.
- K. Otori, Y. Oda, K. Sugiyama, et al., “High frequency of K-ras mutations in human colorectal hyperplastic polyps,” Gut, vol. 40, no. 5, pp. 660–663, 1997.
- K. Forrester, C. Almoguera, and K. Han, “Detection of high incidence of K-ras oncogenes during human colon tumorigenesis,” Nature, vol. 327, no. 6120, pp. 298–303, 1987.
- B. Vogelstein, E. R. Fearon, S. R. Hamilton, et al., “Genetic alterations during colorectal-tumor development,” The New England Journal of Medicine, vol. 319, no. 9, pp. 525–532, 1988.
- K. Kimura, I. Nagasaka, N. Hoshizima, et al., “No duplicate KRAS mutation is identified on the same allele in gastric or colorectal cancer cells with multiple KRAS mutations,” Journal of International Medical Research, vol. 35, no. 4, pp. 450–457, 2007.
- F. Al-Mulla, J. J. Going, E. T. H. H. Sowden, A. Winter, I. R. Pickford, and G. D. Birnie, “Heterogeneity of mutant versus wild-type Ki-ras in primary and metastatic colorectal carcinomas, and association of codon-12 valine with early mortality,” Journal of Pathology, vol. 185, no. 2, pp. 130–138, 1998.
- W. S. Samowitz, K. Curtin, D. Schaffer, M. Robertson, M. Leppert, and M. L. Slattery, “Relationship of Ki-ras mutations in colon cancers to tumor location, stage, and survival: a population-based study,” Cancer Epidemiology Biomarkers and Prevention, vol. 9, no. 11, pp. 1193–1197, 2000.
- J.-I. Park, C. J. Strock, D. W. Ball, and B. D. Nelkin, “The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine-paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway,” Molecular and Cellular Biology, vol. 23, no. 2, pp. 543–554, 2003.
- S. A. Ahrendt, P. A. Decker, E. A. Alawi, et al., “Cigarette smoking is strongly associated with mutation of the K-ras gene in patients with primary adenocarcinoma of the lung,” Cancer, vol. 92, no. 6, pp. 1525–1530, 2001.
- P. Broermann, K. Junker, B. H. Brandt, et al., “Trimodality treatment in stage III nonsmall cell lung carcinoma: prognostic impact of K-ras mutations after neoadjuvant therapy,” Cancer, vol. 94, no. 7, pp. 2055–2062, 2002.
- M. Huncharek, J. Muscat, and J.-F. Geschwind, “K-ras oncogene mutation as a prognostic marker in non-small cell lung cancer: a combined analysis of 881 cases,” Carcinogenesis, vol. 20, no. 8, pp. 1507–1510, 1999.
- W. H. Westra, R. J. C. Slebos, G. J. A. Offerhaus, et al., “K-ras oncogene activation in lung adenocarcinomas from former smokers: evidence that K-ras mutations are an early and irreversible event in the development of adenocarcinoma of the lung,” Cancer, vol. 72, no. 2, pp. 432–438, 1993.
- D. A. Eberhard, B. E. Johnson, L. C. Amler, et al., “Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib,” Journal of Clinical Oncology, vol. 23, no. 25, pp. 5900–5909, 2005.
- A. Hollestelle, F. Elstrodt, J. H. A. Nagel, W. W. Kallemeijn, and M. Schutte, “Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines,” Molecular Cancer Research, vol. 5, no. 2, pp. 195–201, 2007.
- S. Bamford, E. Dawson, S. Forbes, et al., “The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website,” British Journal of Cancer, vol. 91, no. 2, pp. 355–358, 2004.
- I. Uberall, Z. Kolar, R. Trojanec, et al., “The status and role of ErbB receptors in human cancer,” Experimental and Molecular Pathology, vol. 84, no. 2, pp. 79–89, 2008.
- C. L. Hann and J. R. Brahmer, “Who should receive epidermal growth factor receptor inhibitors for non-small cell lung cancer and when?” Current Treatment Options in Oncology, vol. 8, no. 1, pp. 28–37, 2007.
- K. Sugio, H. Uramoto, K. Ono, et al., “Mutations within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking,” British Journal of Cancer, vol. 94, no. 6, pp. 896–903, 2006.
- I. Y. S. Tam, L. P. Chung, W. S. Suen, et al., “Distinct epidermal growth factor receptor and KRAS mutation patterns in non-small cell lung cancer patients with different tobacco exposure and clinicopathologic features,” Clinical Cancer Research, vol. 12, no. 5, pp. 1647–1653, 2006.
- K. Tamura and M. Fukuoka, “Gefitinib in non-small cell lung cancer,” Expert Opinion on Pharmacotherapy, vol. 6, no. 6, pp. 985–993, 2005.
- K. Hotta, K. Kiura, H. Ueoka, et al., “Effect of gefitinib (‘Iressa’, ZD1839) on brain metastases in patients with advanced non-small-cell lung cancer,” Lung Cancer, vol. 46, no. 2, pp. 255–261, 2004.
- J. A. Katzel, M. P. Fanucchi, and Z. Li, “Recent advances of novel targeted therapy in non-small cell Lung cancer,” Journal of Hematology and Oncology, vol. 2, article 2, 2009.
- G. D. Demetri, “Targeting c-kit mutations in solid tumors: scientific rationale and novel therapeutic options,” Seminars in Oncology, vol. 28, no. 5, supplement 17, pp. 19–26, 2001.
- D. J. L. Joske, “Chronic myeloid leukaemia: the evolution of gene-targeted therapy,” Medical Journal of Australia, vol. 189, no. 5, pp. 277–282, 2008.
- A. Agarwal, C. A. Eide, A. Harlow, et al., “An activating KRAS mutation in imatinib-resistant chronic myeloid leukemia,” Leukemia, vol. 22, no. 12, pp. 2269–2272, 2008.
- A. Lievre, J.-B. Bachet, V. Boige, et al., “KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab,” Journal of Clinical Oncology, vol. 26, no. 3, pp. 374–379, 2008.
- R. M. Giusti, K. Shastri, A. M. Pilaro, et al., “U.S. food and drug administration approval: panitumumab for epidermal growth factor receptor-expressing metastatic colorectal carcinoma with progression following fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens,” Clinical Cancer Research, vol. 14, no. 5, pp. 1296–1302, 2008.
- R. G. Amado, M. Wolf, M. Peeters, et al., “Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer,” Journal of Clinical Oncology, vol. 26, no. 10, pp. 1626–1634, 2008.
- T. M. Grana, E. V. Rusyn, H. Zhou, C. I. Sartor, and A. D. Cox, “Ras mediates radioresistance through both phosphatidylinositol 3-kinase-dependent and Raf-dependent but mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-independent signaling pathways,” Cancer Research, vol. 62, no. 14, pp. 4142–4150, 2002.
- J. M. Escandell, P. Kaler, M. C. Recio, et al., “Activated KRAS protects colon cancer cells from cucurbitacin-induced apoptosis: the role of p53 and p21,” Biochemical Pharmacology, vol. 76, no. 2, pp. 198–207, 2008.
- V. Samouelian, C. M. Maugard, M. Jolicoeur, et al., “Chemosensitivity and radiosensitivity profiles of four new human epithelial ovarian cancer cell lines exhibiting genetic alterations in BRCA2, TGF-RII, KRAS2, TP53 and/or CDNK2A,” Cancer Chemotherapy and Pharmacology, vol. 54, no. 6, pp. 497–504, 2004.
- R. Rosell, J. L. Gonzalez-Larriba, V. Alberola, et al., “Single-agent paclitaxel by 3-hour infusion in the treatment of non-small cell lung cancer: links between p53 and K-ras gene status and chemosensitivity,” Seminars in Oncology, vol. 22, no. 6, supplement 14, pp. 12–18, 1995.
- G. W. Jean and S. R. Shah, “Epidermal growth factor receptor monoclonal antibodies for the treatment of metastatic colorectal cancer,” Pharmacotherapy, vol. 28, no. 6, pp. 742–754, 2008.
- I. Sirak, J. Petera, J. Hatlova, et al., “Epidermal growth factor receptor as a predictor of tumor response to preoperative chemoradiation in locally advanced gastric carcinoma,” Strahlentherapie und Onkologie, vol. 184, no. 11, pp. 592–597, 2008.
- P. J. Ross, M. George, D. Cunningham, et al., “Inhibition of Kirsten-ras expression in human colorectal cancer using rationally selected Kirsten-ras antisense oligonucleotides,” Molecular Cancer Therapeutics, vol. 1, no. 1, pp. 29–41, 2001.
- N. Ikeda, Y. Nakajima, M. Sho, et al., “The association of K-ras gene mutation and vascular endothelial growth factor gene expression in pancreatic carcinoma,” Cancer, vol. 92, no. 3, pp. 488–499, 2001.
- T. Konishi, C. L. Huang, M. Adachi, et al., “The K-ras gene regulates vascular endothelial growth factor gene expression in non-small cell lung cancers,” International Journal of Oncology, vol. 16, no. 3, pp. 501–511, 2000.
- H. Hurwitz and S. Saini, “Bevacizumab in the treatment of metastatic colorectal cancer: safety profile and management of adverse events,” Seminars in Oncology, vol. 33, supplement 10, pp. S26–S34, 2006.
- Z. Zhang, G. Jiang, F. Yang, and J. Wang, “Knockdown of mutant K-ras expression by adenovirus-mediated siRNA inhibits the in vitro and in vivo growth of lung cancer cells,” Cancer Biology & Therapy, vol. 5, no. 11, pp. 1481–1486, 2006.
- A. W. Tong, Y.-A. Zhang, C. Cunningham, P. Maples, and J. Nemunaitis, “Potential clinical application of antioncogene ribozymes for human lung cancer,” Clinical Lung Cancer, vol. 2, no. 3, pp. 220–226, 2001.
- N. M. G. M. Appels, J. H. Beijnen, and J. H. M. Schellens, “Development of farnesyl transferase inhibitors: a review,” Oncologist, vol. 10, no. 8, pp. 565–578, 2005.
- S. F. Sousa, P. A. Fernandes, and M. J. Ramos, “Farnesyltransferase inhibitors: a detailed chemical view on an elusive biological problem,” Current Medicinal Chemistry, vol. 15, no. 15, pp. 1478–1492, 2008.