REVIEW URRENT C OPINION
Ras in digestive oncology: from molecular biology to clinical implications Nicolas Charette a, Caroline Vandeputte a, and Peter Sta¨rkel b
Purpose of review The modalities of Ras mutation detection, its role as a predictive biomarker, mechanisms of wild-type Ras activation, and the role of Ras-directed targeted therapies will be discussed mainly in colorectal cancer. Recent findings RAS genotype is generally considered to be highly concordant between primary colorectal tumours and metastases. However, recent data show significant discordance between primary tumours and specific metastatic sites, but also heterogeneity within primary tumours. Moreover, the mechanisms of Ras activation expand far beyond mutations through altered expression or function of physiological Ras activators and inhibitors. Accordingly, genomic signatures of Ras or epidermal growth factor receptor (EGFR) activation are being developed and are potential predictive biomarkers of response to anti-EGFR antibodies. Finally, several recent clinical trials targeting Ras or its downstream signalling with statins or Raf inhibitors have shown promising activity in chemorefractory metastatic colorectal cancer. Summary RAS mutation remains an important biomarker predicting response to anti-EGFR therapies and perhaps clinical outcomes after surgery for metastatic colorectal cancer, but new techniques including genomic signatures need to be validated to take into account the complexity of Ras activation. The importance of Ras signalling as a therapeutic target has recently been outlined by successful clinical trials with Raf inhibitors. Keywords colorectal cancer, Raf, Ras, targeted therapies
INTRODUCTION Ras is a family of four highly homogeneous proteins (H-Ras, N-Ras, K-Ras4A and K-Ras4B) encoded by three ubiquitously expressed genes (HRAS, NRAS, and KRAS) [1]. They are molecular switches that transduce extracellular signals to the cytosol and the nucleus, promoting cellular proliferation, resistance to apoptosis, genomic instability, cell migration, angiogenesis and impairing antitumour immune response [2]. All of these biological functions are hallmarks of cancer [3]. Accordingly, Ras mutations are found in 16% of human tumours, in which they typically consist of point mutations in three hotspots in codons 12, 13 and 61 [4 ]. According to the Catalogue of Somatic Mutations in Cancer database [5], Ras mutations are particularly prevalent in digestive malignancies (Table 1).
control of Ras guanine nucleotide exchange factors (RasGEFs) and Ras GTPase-activating proteins (RasGAPs), respectively [6]. The stimulation of cell membrane receptors – including tyrosine kinase receptors [7], antigen receptors [8], cytokine receptors [9] and G-protein-coupled receptors [10] – leads to the translocation of cytosolic RasGEFs to the plasma membrane where they activate membranebound Ras. Similarly, RasGAP-mediated inactivation of Ras requires translocation of the protein to the cell membrane, formation of a complex with Ras and stimulation of its weak intrinsic GTPase
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REGULATION OF RAS ACTIVATION Ras proteins alternate between an active, guanosine triphosphate (GTP)-bound, state and an inactive, guanosine diphosphate-bound, state, under the www.co-oncology.com
a Department of Medicine, Digestive Oncology Unit, Institut Jules Bordet, Universite´ Libre de Bruxelles and bDepartment of Gastroenterology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
Correspondence to Nicolas Charette, Department of Medicine, Digestive Oncology Unit and Nutrition Team, Institut Jules Bordet, Rue He´gerBordet 1, 1000 Brussels, Belgium. Tel: +32 2 5413196; e-mail: nicolas. [email protected] Curr Opin Oncol 2014, 26:454–461 DOI:10.1097/CCO.0000000000000088 Volume 26 Number 4 July 2014
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Ras in digestive oncology Charette et al.
KEY POINTS Activation of Ras is frequently observed in gastrointestinal cancers regardless of the presence of RAS mutations. Substantial intratumoral genomic heterogeneity may lead to misclassification of RAS mutation status, which may be prevented by analysis of free circulating tumour DNA. KRAS and NRAS mutations are currently the only valid predictors of nonresponse to anti-EGFR antibodies in colorectal cancer, but Ras-related genomic signatures are likely to be better predictors of response to these therapies. KRAS mutation is a prognostic factor that predicts shorter overall survival after resection of liver metastasis in colorectal cancer. Agents targeting Raf, that is sorafenib and regorafenib, show modest clinical efficacy in chemorefractory metastatic colorectal cancer.
activity [11]. The most common somatic mutations responsible for Ras activation in cancer impair the Ras-RasGAP complex formation or activity [12], leading to a reduced GTPase activity [13]. Activated Ras needs to interact with scaffold proteins such as galectins to stabilize its association with the cell membrane and to activate downstream signalling [14,15].
RAS DOWNSTREAM SIGNALLING The pleiotropic biological effects of Ras are mediated by multiple downstream proteins sharing a recognition domain for active Ras [16] (Fig. 1). These Ras effectors activate numerous intracellular signalling pathways acting in complex integrated networks characterized by multiple crosstalks as well as negative and positive feedback loops [17 ]. &&
Table 1. Prevalence of Ras mutations in digestive cancer Tumour type
K-Ras (%)
H-Ras (%)
N-Ras (%)
Biliary tract
25.2
0.0
2.1
Large intestine
34.7
0.5
3.9
Liver
3.0
0.3
1.0
Oesophagus
2.3
0.7
0.0 0.8
Pancreas
57.3
0.0
Peritoneum
28.3
0.0
ND
Small intestine
19.5
0.0
0.0
6.0
2.0
1.2
Stomach
ND, not determined. Data from the Catalogue of Somatic Mutations in Cancer v68 database of the Wellcome Trust Sanger Institute, Cambridge, UK (http:// www.sanger.ac.uk/cosmic).
Raf family members (Raf-1, B-Raf and A-Raf) were the first Ras effectors to be identified [18]. Activation of Raf is initiated by recruitment to the cell membrane by GTP-bound Ras, which is followed by a complex and not fully understood process, involving dephosphorylation, phosphorylation by several membrane-resident kinases and oligomerization [19]. Raf then activates mitogen-activated protein kinase/ERK kinase (MEK)1/2, which in turn induces activation of extracellular signal regulated kinase (ERK)1/2. ERK has more than 160 identified cytosolic and nuclear targets, and promotes cell proliferation, survival, growth and motility [20]. The importance of Ras for Raf activation and that of Raf in Ras downstream signalling is highlighted by the fact that Ras and Raf mutations are mutually exclusive in human tumours [21]. The second Ras effector that has been identified was PI3Kinase [22]. Binding of Ras to the p110 catalytic subunit of the class I PI3Kinase leads to increased translocation of this kinase to the cell membrane [23]. However, activation of PI3kinase also requires the binding of its p85 regulatory subunit to phosphotyrosine residues of activated tyrosine kinase receptors [24]. The relative role of Ras and additional stimuli in PI3Kinase activation may vary in different cell types. Indeed, Ras and PI3kinase mutations are mutually exclusive in some cancer types, such as endometrial cancers, but do coexist in others, such as colorectal cancer [25]. Translocation and activation of PI3Kinase results in accumulation of PI(3,4,5)P3 in the cell membrane [26], which serves as a docking site for 3-phosphoinositide dependent protein kinase-1 (PDK1) and Akt. Akt is then activated upon phosphorylation by PDK1 and the mammalian target of rapamycin (mTOR)–rictor complex [27,28]. Akt modulates the activity of more than 100 substrates, including mTOR, leading to increased cell survival, cell growth and cell proliferation, to promotion of angiogenesis and cell migration and to regulation of glucose and lipid metabolism [29]. Along with Raf and PI3Kinase, Ras activates several other effectors including RalGDS [30], PLCe [31] and TIAM1 [32], all of which exhibit oncogenic properties. Importantly, interaction of Ras with several of its binding partners, such as the Ras association domain-containing family (RASSF) family members, leads to tumour suppression through induction of apoptosis [33].
MECHANISMS OF WILD-TYPE RAS ACTIVATION In addition to mutations, many other mechanisms may lead to Ras activation, as is the case in hepatocarcinoma. Indeed, direct evaluation of GTP-bound
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RasGDP GEFs
GAPs
RasGTP Growth factor receptor
AF–6
TIAM1
PI3K
RGS
Rain
Rin1
Raf
Ral Apoptosis BAD
ASK
MST
Rok
ERK GSK-3β mTOR NF-κB
Proliferation, survival, endo- & exocytosis
FOXO Cytoplasmic substrates
Proliferation, survival, motility
RalGDS
PLCε
MEK
Akt Rac ↓ Ras, Notch & Wnt signaling
RASSF
Proliferation, survival, growth, motility, metabolism, angiogenesis
Survival
Nuclear substrates
Proliferation, survival, growth, motility
Angiogenesis, inflammation
FIGURE 1. Ras effectors and their biological responses. Ras-GTP recruits a wide variety of effectors (white ovals) activating intracellular signalling pathways (white rectangles) leading to cellular processes (grey rectangles) that are important in oncogenesis. Some Ras effectors exhibit tumour-suppressive activities (dark grey ovals). Grey arrows indicate activation, grey T arrows inhibition. Akt stimulates mTOR by inactivating mTOR inhibitors. Black arrows link signalling pathways to their cellular outcome. Dotted arrows indicate poorly understood downstream signalling. AF-6, acute lymphoblastic leukaemia-1 fused gene on chromosome 6; ERK, extracellular signal regulated kinase; GDP, guanosine diphosphate; GF, growth factors; GSK-3b, glycogen synthase kinase-3; GTP, guanosine triphosphate; MEK, mitogen-activated protein kinase/ERK kinase; mTOR, mammalian target of rapamycin; NF-kB, nuclear factor-kB; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; RalGDS, Ral guanine nucleotide dissociation stimulator; RASSF, Ras association domain-containing family; RGS, regulator of G-protein signalling; Rin1, Ras interaction/interference protein-1; RTK, tyrosine kinase receptor; TIAM1, T-cell lymphoma invasion and metastasis-1.
Ras in resected hepatocarcinomas showed that Ras is more active in the tumour than in surrounding liver and that Ras activation was associated with poor prognosis. Similar results were found when activation of ERK and Akt were assessed in the same patients. Moreover, activation of RalA, another Ras effector was only seen in neoplastic tissue and strongly correlated with prognosis [34]. Ras mutations are uncommon in hepatocarcinoma. By contrast, overexpression of growth factors – such as insulin-like growth factor, hepatocyte growth factor and epidermal growth factor family members – and their receptors participates in Ras activation in hepatocarcinoma [35]. Additionally, downregulation of at least one RasGAP among NF1, DAB2IP and RASAL1 is found in all hepatocarcinoma, which results in sustained Ras activation [36]. Finally, qualitative changes in Ras downstream signalling are also consistently found. Indeed, Ras-induced cell death is impaired in the vast majority of hepatocarcinomas through suppression of at least one of the RASSF family members [37] and levels of Spreds, 456
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physiological inhibitors of the Raf/ERK pathway, are frequently decreased [38]. Recent literature also supports mutation-independent Ras activation in colorectal cancer. First, Her2, a tyrosine kinase receptor known to activate Ras, is overexpressed in minor clones of 61% of patients harbouring a KRAS wild-type tumour, and gene amplification is found in all neoplastic cells in an additional 4% of patients [39 ]. Additionally, amplification of wild-type KRAS occurs in a small fraction of colorectal cancer and has the same clinical consequences as mutations [40 ]. More importantly, recent data strongly suggest that reduced expression or dysfunction of RasGAPs plays a role in colorectal carcinogenesis and may contribute to Ras activation. Epidemiological data show that neurofibromatosis, a genetic disorder characterized by loss of the RasGAP neurofibromin, increases the risk of digestive cancers with hazard ratios of 2.0 for colon, 2.8 for gastric, 3.3 for oesophageal and 3.8 for liver cancer [41 ]. The expression of RASAL1, another RasGAP family member, is &
&
&
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Ras in digestive oncology Charette et al.
reduced through gene promoter methylation in one half of colon adenocarcinomas, one-fifth of large adenomas but not in small adenomas. Reduced RASAL1 expression was mainly found in KRAS wild-type tumours and was associated with Ras activation in vitro [42]. More recently, RASA1, another RasGAP, was found to be downregulated as a consequence of miR-31 overexpression in patients’ samples. Inhibition of RASA1 expression in experimental models led to activation of Ras [43 ]. Finally, qualitative changes are found in Ras downstream signalling that may shift the balance towards a growth promoting effect. Indeed, downregulation of RASSF1A because of methylation was evaluated in three studies, with a prevalence ranging from 14 to 73.5%, and was more frequent in KRAS wild-type tumours [44,45,46 ]. Altogether, these results show that Ras activation is a complex phenomenon that cannot be fully understood by focusing only on Ras mutations (Fig. 2). &
&
EVALUATION OF RAS MUTATION It is generally accepted that mutations of KRAS occur early in colorectal carcinogenesis and remain stable during the course of the disease. According to a recent meta-analysis that included 1157 primary tumours and corresponding metastases, the KRAS genotype was highly concordant in paired samples except between primary tumours and metastatic lymph nodes [47 ]. In addition to this discrepancy &
Growth factors
involving lymph node metastases, another recent study found a 32% discordance rate between primary tumours and lung metastases [48 ]. A recent study in renal carcinoma revealed that intratumoral genomic heterogeneity leads to underestimation of the mutational burden as assessed by single tumour-biopsy samples [49 ]. Based on this seminal work, two research groups analysed KRAS mutations in several parts of resected colon adenocarcinomas. Both found coexistence of KRAS mutated and KRAS wild-type areas [50,51 ]. One of these studies could determine that 44% of the examined samples were heterogeneous regarding KRAS genotype [50]. One way to address this issue could be to assess Ras activation with a phosphorylated ERK immunohistochemical staining, allowing to observe its spatial distribution within the tumour. Unfortunately, phospho-ERK expression does not correlate with KRAS mutations in pancreas [52] or in colorectal adenocarcinoma [53]. Another solution would be to rely on ‘liquid biopsies’. Testing circulating tumour cells of colorectal cancer patients for KRAS mutations is extremely challenging but possible, although the technique still needs to be improved [54]. By contrast, mutant circulating cell free DNA was found in all KRAS mutated patients in one study [55 ]. Moreover, by serially analysing plasma samples of patients treated with anti-epidermal growth factor receptor (EGFR) antibodies for circulating tumour DNA, two groups observed the emergence of KRAS mutations several &
&&
&
&
Increased expression of IGF-2, HB-EGF, TGFα, amphiregulin
RasGDP
Growth factor receptor
SOS
GAPs
Reduced expression of NF1, DAB2IP and RASAL1, RASA1
RasGTP
Increased expression of EGFR, HER2 and c-Met
Raf
MEK
Reduced expression of Spred-1 and 2
Spred1/2
ERK
FIGURE 2. Potential mechanisms of Ras activation in digestive cancers. In addition to RAS and Raf mutation, imbalance between activators (increased expression or activating mutation of growth factors and growth factor receptors) and inhibitors (reduced expression or inactivating mutation of RasGAPs and Spreds) results in unrestrained Ras signalling. Examples of alterations described in digestive cancers are outlined. EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; IGF, insulin-like growth factor; MEK, mitogen-activated protein kinase kinase. 1040-8746 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
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months before evidence of tumour progression [56,57 ]. These results provide a good example of the potential clinical utility of circulating tumour DNA analyses. &&
RAS AS PREDICTIVE BIOMARKER Ras is known by digestive oncologists largely because of the role of KRAS mutations in predicting nonresponse to anti-EGFR antibodies [58,59]. However, most patients with wild-type KRAS do not respond to these treatments. To refine patient selection, less frequent RAS mutations have been studied. One recent study showed that NRAS mutations predict resistance to panitumumab [60 ], which paralleled the association between NRAS mutations and resistance to cetuximab [61]. As a consequence, it is now widely accepted that patients harbouring such mutations should not receive anti-EGFR antibodies. The role of additional biomarkers, in particular BRAF and PIK3CA mutations and loss of phosphatase and tensin homolog expression, is still debated. A recent meta-analysis of 20 studies including 1773 patients found that these three biomarkers where associated with poor clinical outcomes in metastatic KRAS wild-type colorectal cancer treated with antiEGFR antibodies. However, these data are largely based on poor quality evidence and their clinical relevance remains unclear [62]. Furthermore, a more recent retrospective study including 572 patients found that they did not predict benefit from cetuximab [63]. Finally, two genomic signatures allowing the identification of colorectal cancer with an active Ras [64] or EGFR-signalling pathway [65 ] have been developed. Both predict response to anti-EGFR treatment better than KRAS mutation status, but also identify potential candidates for downstream pathway inhibition. Recently, Ras status has been found to be a prognostic biomarker in patients undergoing liver resection for colorectal cancer metastases. Indeed, four independent groups observed that KRAS mutations independently predict lower disease-specific survival [66] or lower recurrence-free and overall survival [67,68,69 ]. One of these studies found a higher rate of lung recurrence after resection of KRAS mutated liver metastases [69 ]. These data are in line with the observation that KRAS mutations are associated with lung and perhaps brain relapse after curative resection of a primary colorectal cancer [70]. &
&
&&
&&
RAS AS A THERAPEUTIC TARGET To date, no successful specific Ras inhibitor has reached clinical practice. Ras proteins bind guanine nucleotides with a very high affinity, in the 458
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nanomolar range [71], whereas cellular concentrations of GTP are in the millimolar range [72]. As a consequence, reducing this affinity by a factor of 100 does not prevent Ras from binding GTP [71] and therapeutic attempts aiming to reduce Ras GTP loading are likely to be unsuccessful. Accordingly, efforts have focused on indirect approaches to inhibit Ras function by targeting the regulation of Ras membrane localization or by inhibition of downstream signalling pathways. Another approach that theoretically allows to specifically target Ras-mutated tumours is based on the model of synthetic lethality. According to this concept, two genes are synthetically lethal if loss of function of either of them alone does not impair cell viability, whereas loss of activity of both of them leads to cell death [73]. Approaches based on this theory are currently explored in the preclinical setting [74,75]. Ras transforming activity and its ability to transduce extracellular signals depends upon its association with cellular membranes. Targeting of Ras to cell membranes is mediated by several posttranslational modifications involving its C-terminal domain that sequentially undergoes three major modifications: prenylation, proteolysis and methylation [76]. Prenylation, which consists of the addition of a lipid moiety, generally a farnesyl and occasionally a geranylgeranyl isoprenoid, is the crucial step in membrane targeting of Ras [77]. Farnesyl pyrophosphate, the lipid donor in the farnesylation reaction, is an intermediate component of the mevalonate pathway. Statins, by inhibiting the rate-limiting reaction of this biosynthetic pathway, could theoretically inhibit farnesylation. Unfortunately, in the usual conditions of treatment that effectively reduce cholesterol synthesis, there is no observable effect on protein farnesylation [78]. Nevertheless, a phase II clinical trial assessing the potential of high-dose simvastatin to overcome cetuximab resistance in KRAS mutant chemorefractory colorectal cancer showed a promising median progression-free and overall survival of 7.6 and 12.8 months, respectively, in patients treated with simvastatin, cetuximab and irinotecan [79 ]. On the contrary, despite promising preclinical data, two farnesyltransferase inhibitors, lonafarnib and tipifarnib, have been evaluated in phase III clinical trials but failed to increase overall survival in pancreatic and colon adenocarcinoma [80,81]. Salirasib is an S-farnesyl cysteine analogue that dislodges active Ras from the cell membrane by competition for membrane docking sites, thereby inhibiting Ras downstream signalling [82]. This is followed by an accelerated degradation of cytosolic Ras and a reduction of the total amount of cellular Ras [83]. A phase I study was performed in &&
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association with gemcitabine in 19 patients with metastatic pancreas adenocarcinoma. This study showed that the association is well tolerated and results in a 1-year survival rate of 37% [84 ]. Preclinical studies also suggest a potential benefit of this compound in other types of cancer such as hepatocarcinoma [85,86] and colon adenocarcinoma [87]. Numerous drugs targeting the Raf-MEK-ERK and the PI3Kinase-Akt-mTOR pathways are under preclinical or early clinical development [88 ]. In the field of digestive oncology, 2013 has seen the emergence of Raf as a relevant target. Indeed, a phase III trial assessing regorafenib, a multikinase inhibitor that potently inhibits Raf among others, in patients with chemorefractory metastatic colorectal cancer found an increased median overall survival with a hazard ratio of 0.77. However, although the trial has been considered as positive, it translated into a disappointing increase in median overall survival from 5.0 to only 6.4 months [89 ]. Furthermore, a phase I/II trial testing sorafenib in combination with irinotecan in patients with KRAS mutant chemorefractory metastatic colorectal cancers found a promising activity of this combination [90 ]. &
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CONCLUSION Ras activation and downstream signalling is a highly sophisticated process, integrating a broad range of extracellular signals and involving numerous regulatory proteins. As a consequence, individual mutation or deregulation of several different proteins may lead to Ras activation and looking only at mutations of RAS or a few well known Ras partners does not allow to fully appreciate the extent of Ras activation. Although RAS mutations are useful predictive markers of response to anti-EGFR antibodies, the development of Ras-related genomic signatures will probably better identify responders to such treatments. Additionally, drugs targeting Raf, such as regorafenib and sorafenib, have shown modest clinical activity in metastatic colorectal cancer but are associated with substantial toxicity. Therefore, it is of paramount importance to predict response or nonresponse to these drugs, either with genomic signatures prior to treatment or with multimodality imaging performed early after treatment initiation, to avoid unnecessary adverse events in this fragile patient population. Acknowledgements None. Conflicts of interest The authors declare no conflict of interest related with this work.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Reuther GW, Der CJ. The Ras branch of small GTPases: Ras family members don’t fall far from the tree. Curr Opin Cell Biol 2000; 12:157–165. 2. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 2011; 11:761–774. 3. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144:646–674. 4. Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in && cancer. Cancer Res 2012; 72:2457–2467. An extensive and up-to-date review of Ras mutations in human cancer, including molecular abnormality underlying the occurrence of these mutations. 5. Bamford S, Dawson E, Forbes S, et al. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br J Cancer 2004; 91:355–358. 6. Malumbres M, Barbacid M. RAS oncogenes the first 30 years. Nat Rev Cancer 2003; 3:459–465. 7. Gschwind A, Fischer OM, Ullrich A. The discovery of receptor tyrosine kinases: targets for cancer therapy. Nat Rev Cancer 2004; 4:361–370. 8. Genot E, Cantrell DA. Ras regulation and function in lymphocytes. Curr Opin Immunol 2000; 12:289–294. 9. Piu F, Magnani M, Ader ME. Dissection of the cytoplasmic domains of cytokine receptors involved in STAT and Ras dependent proliferation. Oncogene 2002; 21:3579–3591. 10. Marinissen MJ, Gutkind JS. G-protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol Sci 2001; 22:368–376. 11. Grewal T, Koese M, Tebar F, Enrich C. Differential regulation of RasGAPs in cancer. Genes Cancer 2011; 2:288–297. 12. Scheffzek K, Ahmadian MR, Kabsch W, et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 1997; 277:333–338. 13. Gibbs JB, Sigal IS, Poe M, Scolnick EM. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci U S A 1984; 81:5704–5708. 14. Shalom-Feuerstein R, Levy R, Makovski V, et al. Galectin-3 regulates RasGRP4-mediated activation of N-Ras and H-Ras. Biochim Biophys Acta 2008; 1783:985–993. 15. Paz A, Haklai R, Elad-Sfadia G, et al. Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation. Oncogene 2001; 20:7486–7493. 16. Herrmann C. Ras-effector interactions: after one decade. Curr Opin Struct Biol 2003; 13:122–129. 17. Aksamitiene E, Kiyatkin A, Kholodenko BN. Cross-talk between mitogenic && Ras/MAPK and survival PI3K/Akt pathways: a fine balance. Biochem Soc Trans 2012; 40:139–146. A complete review of the interaction between the Raf/MAPK and the PI3K/Akt pathway downstream of Ras. This is an excellent introduction to the often overlooked complexity of Ras downstream signalling. 18. Moodie SA, Willumsen BM, Weber MJ, Wolfman A. Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 1993; 260:1658–1661. 19. Matallanas D, Birtwistle M, Romano D, et al. Raf family kinases: old dogs have learned new tricks. Genes Cancer 2011; 2:232–260. 20. Yoon S, Seger R. The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 2006; 24:21–44. 21. Rajagopalan H, Bardelli A, Lengauer C, et al. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002; 418:934. 22. Rodriguez-Viciana P, Warne PH, Dhand R, et al. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 1994; 370:527–532. 23. Gupta S, Ramjaun AR, Haiko P, et al. Binding of ras to phosphoinositide 3kinase p110alpha is required for ras-driven tumorigenesis in mice. Cell 2007; 129:957–968. 24. Jimenez C, Hernandez C, Pimentel B, Carrera AC. The p85 regulatory subunit controls sequential activation of phosphoinositide 3-kinase by Tyr kinases and Ras. J Biol Chem 2002; 277:41556–41562. 25. Yuan TL, Cantley LC. PI3K pathway alterations in cancer: variations on a theme. Oncogene 2008; 27:5497–5510. 26. Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem 1998; 67:481–507. 27. Alessi DR, James SR, Downes CP, et al. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 1997; 7:261–269. 28. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005; 307:1098–1101. 29. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell 2007; 129:1261–1274. 30. Ferro E, Trabalzini L. RalGDS family members couple Ras to Ral signalling and that’s not all. Cell Signal 2010; 22:1804–1810.
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www.co-oncology.com
459
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Gastrointestinal tract 31. Smrcka AV, Brown JH, Holz GG. Role of phospholipase cepsilon in physiological phosphoinositide signaling networks. Cell Signal 2012; 24:1333– 1343. 32. Lambert JM, Lambert QT, Reuther GW, et al. Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism. Nat Cell Biol 2002; 4:621– 625. 33. Overmeyer JH, Maltese WA. Death pathways triggered by activated Ras in cancer cells. Front Biosci (Landmark Ed) 2011; 16:1693–1713. 34. Calvisi DF, Ladu S, Gorden A, et al. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest 2007; 117:2713–2722. 35. Breuhahn K, Longerich T, Schirmacher P. Dysregulation of growth factor signaling in human hepatocellular carcinoma. Oncogene 2006; 25:3787– 3800. 36. Calvisi DF, Ladu S, Conner EA, et al. Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer. J Hepatol 2011; 54:311–319. 37. Calvisi DF, Evert M, Dombrowski F. Pathogenetic and prognostic significance of inactivation of RASSF proteins in human hepatocellular carcinoma. Mol Biol Int 2012; 2012:849874. 38. Yoshida T, Hisamoto T, Akiba J, et al. Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors. Oncogene 2006; 25:6056–6066. 39. Martin V, Landi L, Molinari F, et al. HER2 gene copy number status may & influence clinical efficacy to anti-EGFR monoclonal antibodies in metastatic colorectal cancer patients. Br J Cancer 2013; 108:668–675. Describes the presence of Her2 amplifications in KRAS wild-type colorectal carcinoma and shows a correlation with clinical response to anti-EGFR therapy. 40. Valtorta E, Misale S, Sartore-Bianchi A, et al. KRAS gene amplification in & colorectal cancer and impact on response to EGFR-targeted therapy. Int J Cancer 2013; 133:1259–1265. Shows that KRAS gene amplification rarely occurs in colorectal cancer but is associated with resistance to anti-EGFR treatment in KRAS wild-type tumours. 41. Seminog OO, Goldacre MJ. Risk of benign tumours of nervous system, and of & malignant neoplasms, in people with neurofibromatosis: population-based record-linkage study. Br J Cancer 2013; 108:193–198. This study evaluates the risk of malignancies in neurofibromatosis and shows a higher risk of digestive malignancies in these patients. 42. Ohta M, Seto M, Ijichi H, et al. Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression. Gastroenterology 2009; 136:206–216. 43. Sun D, Yu F, Ma Y, et al. MicroRNA-31 activates the RAS pathway and & functions as an oncogenic microRNA in human colorectal cancer by repressing RAS p21 GTPase activating protein 1 (RASA1). J Biol Chem 2013; 288:9508–9518. Identifies reduced expression of RASA1 as a potential cause for Ras activation in KRAS wild-type tumours. 44. Cao D, Chen Y, Tang Y, et al. Loss of RASSF1A expression in colorectal cancer and its association with K-ras status. Biomed Res Int 2013; 2013:976765. 45. Nilsson TK, Lof-Ohlin ZM, Sun XF. DNA methylation of the p14ARF, RASSF1A and APC1A genes as an independent prognostic factor in colorectal cancer patients. Int J Oncol 2013; 42:127–133. 46. Chen SP, Wu CC, Huang SY, et al. beta-catenin and K-ras mutations and & RASSF1A promoter methylation in Taiwanese colorectal cancer patients. Genet Test Mol Biomarkers 2012; 16:1277–1281. Altogether these works suggest a role of RASSF1A methylation in colon carcinogenesis. 47. Han CB, Li F, Ma JT, Zou HW. Concordant KRAS mutations in primary and & metastatic colorectal cancer tissue specimens: a meta-analysis and systematic review. Cancer Invest 2012; 30:741–747. This study shows a high concordance of KRAS mutation status between primary colorectal tumours and distant metastases. 48. Kim MJ, Lee HS, Kim JH, et al. Different metastatic pattern according to the & KRAS mutational status and site-specific discordance of KRAS status in patients with colorectal cancer. BMC Cancer 2012; 12:347. This study shows a high discordance rate of KRAS genotype between primary and lung metastasis, suggesting intratumour heterogeneity. 49. Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor heterogeneity and && branched evolution revealed by multiregion sequencing. N Engl J Med 2012; 366:883–892. The first study to show the importance of intratumour heterogeneity in solid cancers. 50. Kosmidou V, Oikonomou E, Vlassi M, et al. Tumor heterogeneity revealed by KRAS, BRAF, and PIK3CA pyrosequencing: KRAS and PIK3CA intratumor mutation profile differences and their therapeutic implications. Hum Mutat 2014; 35:329–340. 51. Perez K, Walsh R, Brilliant K, et al. Heterogeneity of colorectal cancer (CRC) & in reference to KRAS proto-oncogene utilizing WAVE technology. Exp Mol Pathol 2013; 95:74–82. These two studies confirm KRAS genotype heterogeneity in colorectal cancer. 52. Yip-Schneider MT, Lin A, Barnard D, et al. Lack of elevated MAP kinase (Erk) activity in pancreatic carcinomas despite oncogenic K-ras expression. Int J Oncol 1999; 15:271–279.
460
www.co-oncology.com
53. Davies JM, Trembath D, Deal AM, et al. Phospho-ERK and AKT status, but not KRAS mutation status, are associated with outcomes in rectal cancer treated with chemoradiotherapy. Radiat Oncol 2011; 6:114. 54. Mostert B, Jiang Y, Sieuwerts AM, et al. KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue. Int J Cancer 2013; 133:130– 141. 55. Mouliere F, El MS, Gongora C, et al. Circulating cell-free DNA from colorectal & cancer patients may reveal high KRAS or BRAF mutation load. Transl Oncol 2013; 6:319–328. This study shows that assessment of KRAS mutations on circulating cell free DNA is feasible and reliable. 56. Diaz LA Jr, Williams RT, Wu J, et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 2012; 486:537–540. 57. Misale S, Yaeger R, Hobor S, et al. Emergence of KRAS mutations and && acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 2012; 486:532–536. These studies show that the emergence of a KRAS mutated subclone undetectable at the time of treatment initiation participates to acquired resistance to antiEGFR therapy. 58. Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008; 26:1626–1634. 59. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 2008; 359:1757–1765. 60. Peeters M, Oliner KS, Parker A, et al. Massively parallel tumor multigene & sequencing to evaluate response to panitumumab in a randomized phase III study of metastatic colorectal cancer. Clin Cancer Res 2013; 19:1902– 1912. Shows that NRAS mutation predicts nonresponse to panitumumab monotherapy. 61. De RW, Claes B, Bernasconi D, et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol 2010; 11:753–762. 62. Yang ZY, Wu XY, Huang YF, et al. Promising biomarkers for predicting the outcomes of patients with KRAS wild-type metastatic colorectal cancer treated with antiepidermal growth factor receptor monoclonal antibodies: a systematic review with meta-analysis. Int J Cancer 2013; 133:1914– 1925. 63. Karapetis CS, Jonker D, Daneshmand M, et al. PIK3CA, BRAF, and PTEN status and benefit from cetuximab in the treatment of advanced colorectal cancer – results from NCIC CTG/AGITG CO.17. Clin Cancer Res 2014; 20:744–753. 64. Guinney J, Ferte C, Dry J, et al. Modeling RAS phenotype in colorectal cancer uncovers novel molecular traits of RAS dependency and improves prediction of response to targeted agents in patients. Clin Cancer Res 2014; 20:265– 272. 65. Tian S, Simon I, Moreno V, et al. A combined oncogenic pathway & signature of BRAF, KRAS and PI3KCA mutation improves colorectal cancer classification and cetuximab treatment prediction. Gut 2013; 62:540–549. These two studies show that genomic signatures allow a better prediction of response to anti-EGFR antibodies than KRAS mutation status. 66. Nash GM, Gimbel M, Shia J, et al. KRAS mutation correlates with accelerated metastatic progression in patients with colorectal liver metastases. Ann Surg Oncol 2010; 17:572–578. 67. Stremitzer S, Stift J, Gruenberger B, et al. KRAS status and outcome of liver resection after neoadjuvant chemotherapy including bevacizumab. Br J Surg 2012; 99:1575–1582. 68. Karagkounis G, Torbenson MS, Daniel HD, et al. Incidence and prognostic impact of KRAS and BRAF mutation in patients undergoing liver surgery for colorectal metastases. Cancer 2013; 119:4137– 4144. 69. Vauthey JN, Zimmitti G, Kopetz SE, et al. RAS mutation status predicts && survival and patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases. Ann Surg 2013; 258:619–626. These studies show that KRAS mutation is a biomarker of adverse prognosis after resection of colorectal liver metastases. 70. Tie J, Lipton L, Desai J, et al. KRAS mutation is associated with lung metastasis in patients with curatively resected colorectal cancer. Clin Cancer Res 2011; 17:1122–1130. 71. Feig LA, Pan BT, Roberts TM, Cooper GM. Isolation of ras GTP-binding mutants using an in situ colony-binding assay. Proc Natl Acad Sci U S A 1986; 83:4607–4611. 72. Traut TW. Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 1994; 140:1–22. 73. Torti D, Trusolino L. Oncogene addiction as a foundational rationale for targeted anticancer therapy: promises and perils. EMBO Mol Med 2011; 3:623–636. 74. Guo W, Wu S, Liu J, Fang B. Identification of a small molecule with synthetic lethality for K-ras and protein kinase C iota. Cancer Res 2008; 68:7403– 7408.
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Ras in digestive oncology Charette et al. 75. Luo J, Emanuele MJ, Li D, et al. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 2009; 137:835– 848. 76. Ahearn IM, Haigis K, Bar-Sagi D, Philips MR. Regulating the regulator: posttranslational modification of RAS. Nat Rev Mol Cell Biol 2012; 13:39–51. 77. Kato K, Cox AD, Hisaka MM, et al. Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc Natl Acad Sci U S A 1992; 89:6403–6407. 78. Sinensky M, Beck LA, Leonard S, Evans R. Differential inhibitory effects of lovastatin on protein isoprenylation and sterol synthesis. J Biol Chem 1990; 265:19937–19941. 79. Lee J, Hong YS, Hong JY, et al. Effect of simvastatin plus cetuximab/irinotecan && for KRAS mutant colorectal cancer and predictive value of the RAS signature for treatment response to cetuximab. Invest New Drugs 2014. [Epub ahead of print] Shows that high-dose simvastatin may reverse cetuximab resistance in KRAS mutant colorectal cancer, with limited toxicity. 80. Van Cutsem E, van de Velde H, Karasek P, et al. Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer. J Clin Oncol 2004; 22:1430–1438. 81. Rao S, Cunningham D, de Gramont A, et al. Phase III double-blind placebocontrolled study of farnesyl transferase inhibitor R115777 in patients with refractory advanced colorectal cancer. J Clin Oncol 2004; 22:3950– 3957. 82. Marom M, Haklai R, Ben-Baruch G, et al. Selective inhibition of Ras-dependent cell growth by farnesylthiosalisylic acid. J Biol Chem 1995; 270:22263– 22270. 83. Haklai R, Weisz MG, Elad G, et al. Dislodgment and accelerated degradation of Ras. Biochemistry 1998; 37:1306–1314.
84. Laheru D, Shah P, Rajeshkumar NV, et al. Integrated preclinical and clinical development of S-trans, trans-farnesylthiosalicylic acid (FTS, Salirasib) in pancreatic cancer. Invest New Drugs 2012; 30:2391–2399. Shows that Ras inhibition is achievable in pancreas cancer and paves the way for further clinical evaluation of salirasib in pancreas adenocarcinoma. 85. Charette N, De SC, Horsmans Y, et al. Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms. Cell Death Dis 2013; 4:e471. 86. Charette N, De Saeger C, Lannoy V, et al. Salirasib inhibits the growth of hepatocarcinoma cell lines in vitro and tumor growth in vivo through ras and mTOR inhibition. Mol Cancer 2010; 9:256. 87. Halaschek-Wiener J, Wacheck V, Schlagbauer-Wadl H, et al. A novel Ras antagonist regulates both oncogenic Ras and the tumor suppressor p53 in colon cancer cells. Mol Med 2000; 6:693–704. 88. McCubrey JA, Steelman LS, Chappell WH, et al. Ras/Raf/MEK/ERK and PI3K/ && PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance. Oncotarget 2012; 3:1068–1111. An extensive review of Ras downstream signalling inhibitors. 89. Grothey A, Van CE, Sobrero A, et al. Regorafenib monotherapy for previously & treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet 2013; 381:303–312. Shows that regorafenib increase overall survival in chemorefractory metastatic colorectal cancer. 90. Samalin E, Bouche O, Thezenas S, et al. Sorafenib and irinotecan (NEXIRI) as & second- or later-line treatment for patients with metastatic colorectal cancer and KRAS-mutated tumours: a multicentre phase I/II trial. Br J Cancer 2014; 110:1148–1154. Shows that sorafenib has promising activity in chemorefractory metastatic colorectal cancer. &
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Ras in digestive oncology: from molecular biology to clinical implications Nicolas Charette a, Caroline Vandeputte a, and Peter Sta¨rkel b
Purpose of review The modalities of Ras mutation detection, its role as a predictive biomarker, mechanisms of wild-type Ras activation, and the role of Ras-directed targeted therapies will be discussed mainly in colorectal cancer. Recent findings RAS genotype is generally considered to be highly concordant between primary colorectal tumours and metastases. However, recent data show significant discordance between primary tumours and specific metastatic sites, but also heterogeneity within primary tumours. Moreover, the mechanisms of Ras activation expand far beyond mutations through altered expression or function of physiological Ras activators and inhibitors. Accordingly, genomic signatures of Ras or epidermal growth factor receptor (EGFR) activation are being developed and are potential predictive biomarkers of response to anti-EGFR antibodies. Finally, several recent clinical trials targeting Ras or its downstream signalling with statins or Raf inhibitors have shown promising activity in chemorefractory metastatic colorectal cancer. Summary RAS mutation remains an important biomarker predicting response to anti-EGFR therapies and perhaps clinical outcomes after surgery for metastatic colorectal cancer, but new techniques including genomic signatures need to be validated to take into account the complexity of Ras activation. The importance of Ras signalling as a therapeutic target has recently been outlined by successful clinical trials with Raf inhibitors. Keywords colorectal cancer, Raf, Ras, targeted therapies
INTRODUCTION Ras is a family of four highly homogeneous proteins (H-Ras, N-Ras, K-Ras4A and K-Ras4B) encoded by three ubiquitously expressed genes (HRAS, NRAS, and KRAS) [1]. They are molecular switches that transduce extracellular signals to the cytosol and the nucleus, promoting cellular proliferation, resistance to apoptosis, genomic instability, cell migration, angiogenesis and impairing antitumour immune response [2]. All of these biological functions are hallmarks of cancer [3]. Accordingly, Ras mutations are found in 16% of human tumours, in which they typically consist of point mutations in three hotspots in codons 12, 13 and 61 [4 ]. According to the Catalogue of Somatic Mutations in Cancer database [5], Ras mutations are particularly prevalent in digestive malignancies (Table 1).
control of Ras guanine nucleotide exchange factors (RasGEFs) and Ras GTPase-activating proteins (RasGAPs), respectively [6]. The stimulation of cell membrane receptors – including tyrosine kinase receptors [7], antigen receptors [8], cytokine receptors [9] and G-protein-coupled receptors [10] – leads to the translocation of cytosolic RasGEFs to the plasma membrane where they activate membranebound Ras. Similarly, RasGAP-mediated inactivation of Ras requires translocation of the protein to the cell membrane, formation of a complex with Ras and stimulation of its weak intrinsic GTPase
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REGULATION OF RAS ACTIVATION Ras proteins alternate between an active, guanosine triphosphate (GTP)-bound, state and an inactive, guanosine diphosphate-bound, state, under the www.co-oncology.com
a Department of Medicine, Digestive Oncology Unit, Institut Jules Bordet, Universite´ Libre de Bruxelles and bDepartment of Gastroenterology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
Correspondence to Nicolas Charette, Department of Medicine, Digestive Oncology Unit and Nutrition Team, Institut Jules Bordet, Rue He´gerBordet 1, 1000 Brussels, Belgium. Tel: +32 2 5413196; e-mail: nicolas. [email protected] Curr Opin Oncol 2014, 26:454–461 DOI:10.1097/CCO.0000000000000088 Volume 26 Number 4 July 2014
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Ras in digestive oncology Charette et al.
KEY POINTS Activation of Ras is frequently observed in gastrointestinal cancers regardless of the presence of RAS mutations. Substantial intratumoral genomic heterogeneity may lead to misclassification of RAS mutation status, which may be prevented by analysis of free circulating tumour DNA. KRAS and NRAS mutations are currently the only valid predictors of nonresponse to anti-EGFR antibodies in colorectal cancer, but Ras-related genomic signatures are likely to be better predictors of response to these therapies. KRAS mutation is a prognostic factor that predicts shorter overall survival after resection of liver metastasis in colorectal cancer. Agents targeting Raf, that is sorafenib and regorafenib, show modest clinical efficacy in chemorefractory metastatic colorectal cancer.
activity [11]. The most common somatic mutations responsible for Ras activation in cancer impair the Ras-RasGAP complex formation or activity [12], leading to a reduced GTPase activity [13]. Activated Ras needs to interact with scaffold proteins such as galectins to stabilize its association with the cell membrane and to activate downstream signalling [14,15].
RAS DOWNSTREAM SIGNALLING The pleiotropic biological effects of Ras are mediated by multiple downstream proteins sharing a recognition domain for active Ras [16] (Fig. 1). These Ras effectors activate numerous intracellular signalling pathways acting in complex integrated networks characterized by multiple crosstalks as well as negative and positive feedback loops [17 ]. &&
Table 1. Prevalence of Ras mutations in digestive cancer Tumour type
K-Ras (%)
H-Ras (%)
N-Ras (%)
Biliary tract
25.2
0.0
2.1
Large intestine
34.7
0.5
3.9
Liver
3.0
0.3
1.0
Oesophagus
2.3
0.7
0.0 0.8
Pancreas
57.3
0.0
Peritoneum
28.3
0.0
ND
Small intestine
19.5
0.0
0.0
6.0
2.0
1.2
Stomach
ND, not determined. Data from the Catalogue of Somatic Mutations in Cancer v68 database of the Wellcome Trust Sanger Institute, Cambridge, UK (http:// www.sanger.ac.uk/cosmic).
Raf family members (Raf-1, B-Raf and A-Raf) were the first Ras effectors to be identified [18]. Activation of Raf is initiated by recruitment to the cell membrane by GTP-bound Ras, which is followed by a complex and not fully understood process, involving dephosphorylation, phosphorylation by several membrane-resident kinases and oligomerization [19]. Raf then activates mitogen-activated protein kinase/ERK kinase (MEK)1/2, which in turn induces activation of extracellular signal regulated kinase (ERK)1/2. ERK has more than 160 identified cytosolic and nuclear targets, and promotes cell proliferation, survival, growth and motility [20]. The importance of Ras for Raf activation and that of Raf in Ras downstream signalling is highlighted by the fact that Ras and Raf mutations are mutually exclusive in human tumours [21]. The second Ras effector that has been identified was PI3Kinase [22]. Binding of Ras to the p110 catalytic subunit of the class I PI3Kinase leads to increased translocation of this kinase to the cell membrane [23]. However, activation of PI3kinase also requires the binding of its p85 regulatory subunit to phosphotyrosine residues of activated tyrosine kinase receptors [24]. The relative role of Ras and additional stimuli in PI3Kinase activation may vary in different cell types. Indeed, Ras and PI3kinase mutations are mutually exclusive in some cancer types, such as endometrial cancers, but do coexist in others, such as colorectal cancer [25]. Translocation and activation of PI3Kinase results in accumulation of PI(3,4,5)P3 in the cell membrane [26], which serves as a docking site for 3-phosphoinositide dependent protein kinase-1 (PDK1) and Akt. Akt is then activated upon phosphorylation by PDK1 and the mammalian target of rapamycin (mTOR)–rictor complex [27,28]. Akt modulates the activity of more than 100 substrates, including mTOR, leading to increased cell survival, cell growth and cell proliferation, to promotion of angiogenesis and cell migration and to regulation of glucose and lipid metabolism [29]. Along with Raf and PI3Kinase, Ras activates several other effectors including RalGDS [30], PLCe [31] and TIAM1 [32], all of which exhibit oncogenic properties. Importantly, interaction of Ras with several of its binding partners, such as the Ras association domain-containing family (RASSF) family members, leads to tumour suppression through induction of apoptosis [33].
MECHANISMS OF WILD-TYPE RAS ACTIVATION In addition to mutations, many other mechanisms may lead to Ras activation, as is the case in hepatocarcinoma. Indeed, direct evaluation of GTP-bound
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RasGDP GEFs
GAPs
RasGTP Growth factor receptor
AF–6
TIAM1
PI3K
RGS
Rain
Rin1
Raf
Ral Apoptosis BAD
ASK
MST
Rok
ERK GSK-3β mTOR NF-κB
Proliferation, survival, endo- & exocytosis
FOXO Cytoplasmic substrates
Proliferation, survival, motility
RalGDS
PLCε
MEK
Akt Rac ↓ Ras, Notch & Wnt signaling
RASSF
Proliferation, survival, growth, motility, metabolism, angiogenesis
Survival
Nuclear substrates
Proliferation, survival, growth, motility
Angiogenesis, inflammation
FIGURE 1. Ras effectors and their biological responses. Ras-GTP recruits a wide variety of effectors (white ovals) activating intracellular signalling pathways (white rectangles) leading to cellular processes (grey rectangles) that are important in oncogenesis. Some Ras effectors exhibit tumour-suppressive activities (dark grey ovals). Grey arrows indicate activation, grey T arrows inhibition. Akt stimulates mTOR by inactivating mTOR inhibitors. Black arrows link signalling pathways to their cellular outcome. Dotted arrows indicate poorly understood downstream signalling. AF-6, acute lymphoblastic leukaemia-1 fused gene on chromosome 6; ERK, extracellular signal regulated kinase; GDP, guanosine diphosphate; GF, growth factors; GSK-3b, glycogen synthase kinase-3; GTP, guanosine triphosphate; MEK, mitogen-activated protein kinase/ERK kinase; mTOR, mammalian target of rapamycin; NF-kB, nuclear factor-kB; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; RalGDS, Ral guanine nucleotide dissociation stimulator; RASSF, Ras association domain-containing family; RGS, regulator of G-protein signalling; Rin1, Ras interaction/interference protein-1; RTK, tyrosine kinase receptor; TIAM1, T-cell lymphoma invasion and metastasis-1.
Ras in resected hepatocarcinomas showed that Ras is more active in the tumour than in surrounding liver and that Ras activation was associated with poor prognosis. Similar results were found when activation of ERK and Akt were assessed in the same patients. Moreover, activation of RalA, another Ras effector was only seen in neoplastic tissue and strongly correlated with prognosis [34]. Ras mutations are uncommon in hepatocarcinoma. By contrast, overexpression of growth factors – such as insulin-like growth factor, hepatocyte growth factor and epidermal growth factor family members – and their receptors participates in Ras activation in hepatocarcinoma [35]. Additionally, downregulation of at least one RasGAP among NF1, DAB2IP and RASAL1 is found in all hepatocarcinoma, which results in sustained Ras activation [36]. Finally, qualitative changes in Ras downstream signalling are also consistently found. Indeed, Ras-induced cell death is impaired in the vast majority of hepatocarcinomas through suppression of at least one of the RASSF family members [37] and levels of Spreds, 456
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physiological inhibitors of the Raf/ERK pathway, are frequently decreased [38]. Recent literature also supports mutation-independent Ras activation in colorectal cancer. First, Her2, a tyrosine kinase receptor known to activate Ras, is overexpressed in minor clones of 61% of patients harbouring a KRAS wild-type tumour, and gene amplification is found in all neoplastic cells in an additional 4% of patients [39 ]. Additionally, amplification of wild-type KRAS occurs in a small fraction of colorectal cancer and has the same clinical consequences as mutations [40 ]. More importantly, recent data strongly suggest that reduced expression or dysfunction of RasGAPs plays a role in colorectal carcinogenesis and may contribute to Ras activation. Epidemiological data show that neurofibromatosis, a genetic disorder characterized by loss of the RasGAP neurofibromin, increases the risk of digestive cancers with hazard ratios of 2.0 for colon, 2.8 for gastric, 3.3 for oesophageal and 3.8 for liver cancer [41 ]. The expression of RASAL1, another RasGAP family member, is &
&
&
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Ras in digestive oncology Charette et al.
reduced through gene promoter methylation in one half of colon adenocarcinomas, one-fifth of large adenomas but not in small adenomas. Reduced RASAL1 expression was mainly found in KRAS wild-type tumours and was associated with Ras activation in vitro [42]. More recently, RASA1, another RasGAP, was found to be downregulated as a consequence of miR-31 overexpression in patients’ samples. Inhibition of RASA1 expression in experimental models led to activation of Ras [43 ]. Finally, qualitative changes are found in Ras downstream signalling that may shift the balance towards a growth promoting effect. Indeed, downregulation of RASSF1A because of methylation was evaluated in three studies, with a prevalence ranging from 14 to 73.5%, and was more frequent in KRAS wild-type tumours [44,45,46 ]. Altogether, these results show that Ras activation is a complex phenomenon that cannot be fully understood by focusing only on Ras mutations (Fig. 2). &
&
EVALUATION OF RAS MUTATION It is generally accepted that mutations of KRAS occur early in colorectal carcinogenesis and remain stable during the course of the disease. According to a recent meta-analysis that included 1157 primary tumours and corresponding metastases, the KRAS genotype was highly concordant in paired samples except between primary tumours and metastatic lymph nodes [47 ]. In addition to this discrepancy &
Growth factors
involving lymph node metastases, another recent study found a 32% discordance rate between primary tumours and lung metastases [48 ]. A recent study in renal carcinoma revealed that intratumoral genomic heterogeneity leads to underestimation of the mutational burden as assessed by single tumour-biopsy samples [49 ]. Based on this seminal work, two research groups analysed KRAS mutations in several parts of resected colon adenocarcinomas. Both found coexistence of KRAS mutated and KRAS wild-type areas [50,51 ]. One of these studies could determine that 44% of the examined samples were heterogeneous regarding KRAS genotype [50]. One way to address this issue could be to assess Ras activation with a phosphorylated ERK immunohistochemical staining, allowing to observe its spatial distribution within the tumour. Unfortunately, phospho-ERK expression does not correlate with KRAS mutations in pancreas [52] or in colorectal adenocarcinoma [53]. Another solution would be to rely on ‘liquid biopsies’. Testing circulating tumour cells of colorectal cancer patients for KRAS mutations is extremely challenging but possible, although the technique still needs to be improved [54]. By contrast, mutant circulating cell free DNA was found in all KRAS mutated patients in one study [55 ]. Moreover, by serially analysing plasma samples of patients treated with anti-epidermal growth factor receptor (EGFR) antibodies for circulating tumour DNA, two groups observed the emergence of KRAS mutations several &
&&
&
&
Increased expression of IGF-2, HB-EGF, TGFα, amphiregulin
RasGDP
Growth factor receptor
SOS
GAPs
Reduced expression of NF1, DAB2IP and RASAL1, RASA1
RasGTP
Increased expression of EGFR, HER2 and c-Met
Raf
MEK
Reduced expression of Spred-1 and 2
Spred1/2
ERK
FIGURE 2. Potential mechanisms of Ras activation in digestive cancers. In addition to RAS and Raf mutation, imbalance between activators (increased expression or activating mutation of growth factors and growth factor receptors) and inhibitors (reduced expression or inactivating mutation of RasGAPs and Spreds) results in unrestrained Ras signalling. Examples of alterations described in digestive cancers are outlined. EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; IGF, insulin-like growth factor; MEK, mitogen-activated protein kinase kinase. 1040-8746 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
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months before evidence of tumour progression [56,57 ]. These results provide a good example of the potential clinical utility of circulating tumour DNA analyses. &&
RAS AS PREDICTIVE BIOMARKER Ras is known by digestive oncologists largely because of the role of KRAS mutations in predicting nonresponse to anti-EGFR antibodies [58,59]. However, most patients with wild-type KRAS do not respond to these treatments. To refine patient selection, less frequent RAS mutations have been studied. One recent study showed that NRAS mutations predict resistance to panitumumab [60 ], which paralleled the association between NRAS mutations and resistance to cetuximab [61]. As a consequence, it is now widely accepted that patients harbouring such mutations should not receive anti-EGFR antibodies. The role of additional biomarkers, in particular BRAF and PIK3CA mutations and loss of phosphatase and tensin homolog expression, is still debated. A recent meta-analysis of 20 studies including 1773 patients found that these three biomarkers where associated with poor clinical outcomes in metastatic KRAS wild-type colorectal cancer treated with antiEGFR antibodies. However, these data are largely based on poor quality evidence and their clinical relevance remains unclear [62]. Furthermore, a more recent retrospective study including 572 patients found that they did not predict benefit from cetuximab [63]. Finally, two genomic signatures allowing the identification of colorectal cancer with an active Ras [64] or EGFR-signalling pathway [65 ] have been developed. Both predict response to anti-EGFR treatment better than KRAS mutation status, but also identify potential candidates for downstream pathway inhibition. Recently, Ras status has been found to be a prognostic biomarker in patients undergoing liver resection for colorectal cancer metastases. Indeed, four independent groups observed that KRAS mutations independently predict lower disease-specific survival [66] or lower recurrence-free and overall survival [67,68,69 ]. One of these studies found a higher rate of lung recurrence after resection of KRAS mutated liver metastases [69 ]. These data are in line with the observation that KRAS mutations are associated with lung and perhaps brain relapse after curative resection of a primary colorectal cancer [70]. &
&
&&
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RAS AS A THERAPEUTIC TARGET To date, no successful specific Ras inhibitor has reached clinical practice. Ras proteins bind guanine nucleotides with a very high affinity, in the 458
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nanomolar range [71], whereas cellular concentrations of GTP are in the millimolar range [72]. As a consequence, reducing this affinity by a factor of 100 does not prevent Ras from binding GTP [71] and therapeutic attempts aiming to reduce Ras GTP loading are likely to be unsuccessful. Accordingly, efforts have focused on indirect approaches to inhibit Ras function by targeting the regulation of Ras membrane localization or by inhibition of downstream signalling pathways. Another approach that theoretically allows to specifically target Ras-mutated tumours is based on the model of synthetic lethality. According to this concept, two genes are synthetically lethal if loss of function of either of them alone does not impair cell viability, whereas loss of activity of both of them leads to cell death [73]. Approaches based on this theory are currently explored in the preclinical setting [74,75]. Ras transforming activity and its ability to transduce extracellular signals depends upon its association with cellular membranes. Targeting of Ras to cell membranes is mediated by several posttranslational modifications involving its C-terminal domain that sequentially undergoes three major modifications: prenylation, proteolysis and methylation [76]. Prenylation, which consists of the addition of a lipid moiety, generally a farnesyl and occasionally a geranylgeranyl isoprenoid, is the crucial step in membrane targeting of Ras [77]. Farnesyl pyrophosphate, the lipid donor in the farnesylation reaction, is an intermediate component of the mevalonate pathway. Statins, by inhibiting the rate-limiting reaction of this biosynthetic pathway, could theoretically inhibit farnesylation. Unfortunately, in the usual conditions of treatment that effectively reduce cholesterol synthesis, there is no observable effect on protein farnesylation [78]. Nevertheless, a phase II clinical trial assessing the potential of high-dose simvastatin to overcome cetuximab resistance in KRAS mutant chemorefractory colorectal cancer showed a promising median progression-free and overall survival of 7.6 and 12.8 months, respectively, in patients treated with simvastatin, cetuximab and irinotecan [79 ]. On the contrary, despite promising preclinical data, two farnesyltransferase inhibitors, lonafarnib and tipifarnib, have been evaluated in phase III clinical trials but failed to increase overall survival in pancreatic and colon adenocarcinoma [80,81]. Salirasib is an S-farnesyl cysteine analogue that dislodges active Ras from the cell membrane by competition for membrane docking sites, thereby inhibiting Ras downstream signalling [82]. This is followed by an accelerated degradation of cytosolic Ras and a reduction of the total amount of cellular Ras [83]. A phase I study was performed in &&
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association with gemcitabine in 19 patients with metastatic pancreas adenocarcinoma. This study showed that the association is well tolerated and results in a 1-year survival rate of 37% [84 ]. Preclinical studies also suggest a potential benefit of this compound in other types of cancer such as hepatocarcinoma [85,86] and colon adenocarcinoma [87]. Numerous drugs targeting the Raf-MEK-ERK and the PI3Kinase-Akt-mTOR pathways are under preclinical or early clinical development [88 ]. In the field of digestive oncology, 2013 has seen the emergence of Raf as a relevant target. Indeed, a phase III trial assessing regorafenib, a multikinase inhibitor that potently inhibits Raf among others, in patients with chemorefractory metastatic colorectal cancer found an increased median overall survival with a hazard ratio of 0.77. However, although the trial has been considered as positive, it translated into a disappointing increase in median overall survival from 5.0 to only 6.4 months [89 ]. Furthermore, a phase I/II trial testing sorafenib in combination with irinotecan in patients with KRAS mutant chemorefractory metastatic colorectal cancers found a promising activity of this combination [90 ]. &
&&
&
&
CONCLUSION Ras activation and downstream signalling is a highly sophisticated process, integrating a broad range of extracellular signals and involving numerous regulatory proteins. As a consequence, individual mutation or deregulation of several different proteins may lead to Ras activation and looking only at mutations of RAS or a few well known Ras partners does not allow to fully appreciate the extent of Ras activation. Although RAS mutations are useful predictive markers of response to anti-EGFR antibodies, the development of Ras-related genomic signatures will probably better identify responders to such treatments. Additionally, drugs targeting Raf, such as regorafenib and sorafenib, have shown modest clinical activity in metastatic colorectal cancer but are associated with substantial toxicity. Therefore, it is of paramount importance to predict response or nonresponse to these drugs, either with genomic signatures prior to treatment or with multimodality imaging performed early after treatment initiation, to avoid unnecessary adverse events in this fragile patient population. Acknowledgements None. Conflicts of interest The authors declare no conflict of interest related with this work.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Reuther GW, Der CJ. The Ras branch of small GTPases: Ras family members don’t fall far from the tree. Curr Opin Cell Biol 2000; 12:157–165. 2. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 2011; 11:761–774. 3. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144:646–674. 4. Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in && cancer. Cancer Res 2012; 72:2457–2467. An extensive and up-to-date review of Ras mutations in human cancer, including molecular abnormality underlying the occurrence of these mutations. 5. Bamford S, Dawson E, Forbes S, et al. The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br J Cancer 2004; 91:355–358. 6. Malumbres M, Barbacid M. RAS oncogenes the first 30 years. Nat Rev Cancer 2003; 3:459–465. 7. Gschwind A, Fischer OM, Ullrich A. The discovery of receptor tyrosine kinases: targets for cancer therapy. Nat Rev Cancer 2004; 4:361–370. 8. Genot E, Cantrell DA. Ras regulation and function in lymphocytes. Curr Opin Immunol 2000; 12:289–294. 9. Piu F, Magnani M, Ader ME. Dissection of the cytoplasmic domains of cytokine receptors involved in STAT and Ras dependent proliferation. Oncogene 2002; 21:3579–3591. 10. Marinissen MJ, Gutkind JS. G-protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol Sci 2001; 22:368–376. 11. Grewal T, Koese M, Tebar F, Enrich C. Differential regulation of RasGAPs in cancer. Genes Cancer 2011; 2:288–297. 12. Scheffzek K, Ahmadian MR, Kabsch W, et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 1997; 277:333–338. 13. Gibbs JB, Sigal IS, Poe M, Scolnick EM. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci U S A 1984; 81:5704–5708. 14. Shalom-Feuerstein R, Levy R, Makovski V, et al. Galectin-3 regulates RasGRP4-mediated activation of N-Ras and H-Ras. Biochim Biophys Acta 2008; 1783:985–993. 15. Paz A, Haklai R, Elad-Sfadia G, et al. Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation. Oncogene 2001; 20:7486–7493. 16. Herrmann C. Ras-effector interactions: after one decade. Curr Opin Struct Biol 2003; 13:122–129. 17. Aksamitiene E, Kiyatkin A, Kholodenko BN. Cross-talk between mitogenic && Ras/MAPK and survival PI3K/Akt pathways: a fine balance. Biochem Soc Trans 2012; 40:139–146. A complete review of the interaction between the Raf/MAPK and the PI3K/Akt pathway downstream of Ras. This is an excellent introduction to the often overlooked complexity of Ras downstream signalling. 18. Moodie SA, Willumsen BM, Weber MJ, Wolfman A. Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 1993; 260:1658–1661. 19. Matallanas D, Birtwistle M, Romano D, et al. Raf family kinases: old dogs have learned new tricks. Genes Cancer 2011; 2:232–260. 20. Yoon S, Seger R. The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 2006; 24:21–44. 21. Rajagopalan H, Bardelli A, Lengauer C, et al. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002; 418:934. 22. Rodriguez-Viciana P, Warne PH, Dhand R, et al. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 1994; 370:527–532. 23. Gupta S, Ramjaun AR, Haiko P, et al. Binding of ras to phosphoinositide 3kinase p110alpha is required for ras-driven tumorigenesis in mice. Cell 2007; 129:957–968. 24. Jimenez C, Hernandez C, Pimentel B, Carrera AC. The p85 regulatory subunit controls sequential activation of phosphoinositide 3-kinase by Tyr kinases and Ras. J Biol Chem 2002; 277:41556–41562. 25. Yuan TL, Cantley LC. PI3K pathway alterations in cancer: variations on a theme. Oncogene 2008; 27:5497–5510. 26. Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem 1998; 67:481–507. 27. Alessi DR, James SR, Downes CP, et al. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 1997; 7:261–269. 28. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005; 307:1098–1101. 29. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell 2007; 129:1261–1274. 30. Ferro E, Trabalzini L. RalGDS family members couple Ras to Ral signalling and that’s not all. Cell Signal 2010; 22:1804–1810.
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www.co-oncology.com
459
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Gastrointestinal tract 31. Smrcka AV, Brown JH, Holz GG. Role of phospholipase cepsilon in physiological phosphoinositide signaling networks. Cell Signal 2012; 24:1333– 1343. 32. Lambert JM, Lambert QT, Reuther GW, et al. Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism. Nat Cell Biol 2002; 4:621– 625. 33. Overmeyer JH, Maltese WA. Death pathways triggered by activated Ras in cancer cells. Front Biosci (Landmark Ed) 2011; 16:1693–1713. 34. Calvisi DF, Ladu S, Gorden A, et al. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest 2007; 117:2713–2722. 35. Breuhahn K, Longerich T, Schirmacher P. Dysregulation of growth factor signaling in human hepatocellular carcinoma. Oncogene 2006; 25:3787– 3800. 36. Calvisi DF, Ladu S, Conner EA, et al. Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer. J Hepatol 2011; 54:311–319. 37. Calvisi DF, Evert M, Dombrowski F. Pathogenetic and prognostic significance of inactivation of RASSF proteins in human hepatocellular carcinoma. Mol Biol Int 2012; 2012:849874. 38. Yoshida T, Hisamoto T, Akiba J, et al. Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors. Oncogene 2006; 25:6056–6066. 39. Martin V, Landi L, Molinari F, et al. HER2 gene copy number status may & influence clinical efficacy to anti-EGFR monoclonal antibodies in metastatic colorectal cancer patients. Br J Cancer 2013; 108:668–675. Describes the presence of Her2 amplifications in KRAS wild-type colorectal carcinoma and shows a correlation with clinical response to anti-EGFR therapy. 40. Valtorta E, Misale S, Sartore-Bianchi A, et al. KRAS gene amplification in & colorectal cancer and impact on response to EGFR-targeted therapy. Int J Cancer 2013; 133:1259–1265. Shows that KRAS gene amplification rarely occurs in colorectal cancer but is associated with resistance to anti-EGFR treatment in KRAS wild-type tumours. 41. Seminog OO, Goldacre MJ. Risk of benign tumours of nervous system, and of & malignant neoplasms, in people with neurofibromatosis: population-based record-linkage study. Br J Cancer 2013; 108:193–198. This study evaluates the risk of malignancies in neurofibromatosis and shows a higher risk of digestive malignancies in these patients. 42. Ohta M, Seto M, Ijichi H, et al. Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression. Gastroenterology 2009; 136:206–216. 43. Sun D, Yu F, Ma Y, et al. MicroRNA-31 activates the RAS pathway and & functions as an oncogenic microRNA in human colorectal cancer by repressing RAS p21 GTPase activating protein 1 (RASA1). J Biol Chem 2013; 288:9508–9518. Identifies reduced expression of RASA1 as a potential cause for Ras activation in KRAS wild-type tumours. 44. Cao D, Chen Y, Tang Y, et al. Loss of RASSF1A expression in colorectal cancer and its association with K-ras status. Biomed Res Int 2013; 2013:976765. 45. Nilsson TK, Lof-Ohlin ZM, Sun XF. DNA methylation of the p14ARF, RASSF1A and APC1A genes as an independent prognostic factor in colorectal cancer patients. Int J Oncol 2013; 42:127–133. 46. Chen SP, Wu CC, Huang SY, et al. beta-catenin and K-ras mutations and & RASSF1A promoter methylation in Taiwanese colorectal cancer patients. Genet Test Mol Biomarkers 2012; 16:1277–1281. Altogether these works suggest a role of RASSF1A methylation in colon carcinogenesis. 47. Han CB, Li F, Ma JT, Zou HW. Concordant KRAS mutations in primary and & metastatic colorectal cancer tissue specimens: a meta-analysis and systematic review. Cancer Invest 2012; 30:741–747. This study shows a high concordance of KRAS mutation status between primary colorectal tumours and distant metastases. 48. Kim MJ, Lee HS, Kim JH, et al. Different metastatic pattern according to the & KRAS mutational status and site-specific discordance of KRAS status in patients with colorectal cancer. BMC Cancer 2012; 12:347. This study shows a high discordance rate of KRAS genotype between primary and lung metastasis, suggesting intratumour heterogeneity. 49. Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor heterogeneity and && branched evolution revealed by multiregion sequencing. N Engl J Med 2012; 366:883–892. The first study to show the importance of intratumour heterogeneity in solid cancers. 50. Kosmidou V, Oikonomou E, Vlassi M, et al. Tumor heterogeneity revealed by KRAS, BRAF, and PIK3CA pyrosequencing: KRAS and PIK3CA intratumor mutation profile differences and their therapeutic implications. Hum Mutat 2014; 35:329–340. 51. Perez K, Walsh R, Brilliant K, et al. Heterogeneity of colorectal cancer (CRC) & in reference to KRAS proto-oncogene utilizing WAVE technology. Exp Mol Pathol 2013; 95:74–82. These two studies confirm KRAS genotype heterogeneity in colorectal cancer. 52. Yip-Schneider MT, Lin A, Barnard D, et al. Lack of elevated MAP kinase (Erk) activity in pancreatic carcinomas despite oncogenic K-ras expression. Int J Oncol 1999; 15:271–279.
460
www.co-oncology.com
53. Davies JM, Trembath D, Deal AM, et al. Phospho-ERK and AKT status, but not KRAS mutation status, are associated with outcomes in rectal cancer treated with chemoradiotherapy. Radiat Oncol 2011; 6:114. 54. Mostert B, Jiang Y, Sieuwerts AM, et al. KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue. Int J Cancer 2013; 133:130– 141. 55. Mouliere F, El MS, Gongora C, et al. Circulating cell-free DNA from colorectal & cancer patients may reveal high KRAS or BRAF mutation load. Transl Oncol 2013; 6:319–328. This study shows that assessment of KRAS mutations on circulating cell free DNA is feasible and reliable. 56. Diaz LA Jr, Williams RT, Wu J, et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 2012; 486:537–540. 57. Misale S, Yaeger R, Hobor S, et al. Emergence of KRAS mutations and && acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 2012; 486:532–536. These studies show that the emergence of a KRAS mutated subclone undetectable at the time of treatment initiation participates to acquired resistance to antiEGFR therapy. 58. Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008; 26:1626–1634. 59. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 2008; 359:1757–1765. 60. Peeters M, Oliner KS, Parker A, et al. Massively parallel tumor multigene & sequencing to evaluate response to panitumumab in a randomized phase III study of metastatic colorectal cancer. Clin Cancer Res 2013; 19:1902– 1912. Shows that NRAS mutation predicts nonresponse to panitumumab monotherapy. 61. De RW, Claes B, Bernasconi D, et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol 2010; 11:753–762. 62. Yang ZY, Wu XY, Huang YF, et al. Promising biomarkers for predicting the outcomes of patients with KRAS wild-type metastatic colorectal cancer treated with antiepidermal growth factor receptor monoclonal antibodies: a systematic review with meta-analysis. Int J Cancer 2013; 133:1914– 1925. 63. Karapetis CS, Jonker D, Daneshmand M, et al. PIK3CA, BRAF, and PTEN status and benefit from cetuximab in the treatment of advanced colorectal cancer – results from NCIC CTG/AGITG CO.17. Clin Cancer Res 2014; 20:744–753. 64. Guinney J, Ferte C, Dry J, et al. Modeling RAS phenotype in colorectal cancer uncovers novel molecular traits of RAS dependency and improves prediction of response to targeted agents in patients. Clin Cancer Res 2014; 20:265– 272. 65. Tian S, Simon I, Moreno V, et al. A combined oncogenic pathway & signature of BRAF, KRAS and PI3KCA mutation improves colorectal cancer classification and cetuximab treatment prediction. Gut 2013; 62:540–549. These two studies show that genomic signatures allow a better prediction of response to anti-EGFR antibodies than KRAS mutation status. 66. Nash GM, Gimbel M, Shia J, et al. KRAS mutation correlates with accelerated metastatic progression in patients with colorectal liver metastases. Ann Surg Oncol 2010; 17:572–578. 67. Stremitzer S, Stift J, Gruenberger B, et al. KRAS status and outcome of liver resection after neoadjuvant chemotherapy including bevacizumab. Br J Surg 2012; 99:1575–1582. 68. Karagkounis G, Torbenson MS, Daniel HD, et al. Incidence and prognostic impact of KRAS and BRAF mutation in patients undergoing liver surgery for colorectal metastases. Cancer 2013; 119:4137– 4144. 69. Vauthey JN, Zimmitti G, Kopetz SE, et al. RAS mutation status predicts && survival and patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases. Ann Surg 2013; 258:619–626. These studies show that KRAS mutation is a biomarker of adverse prognosis after resection of colorectal liver metastases. 70. Tie J, Lipton L, Desai J, et al. KRAS mutation is associated with lung metastasis in patients with curatively resected colorectal cancer. Clin Cancer Res 2011; 17:1122–1130. 71. Feig LA, Pan BT, Roberts TM, Cooper GM. Isolation of ras GTP-binding mutants using an in situ colony-binding assay. Proc Natl Acad Sci U S A 1986; 83:4607–4611. 72. Traut TW. Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 1994; 140:1–22. 73. Torti D, Trusolino L. Oncogene addiction as a foundational rationale for targeted anticancer therapy: promises and perils. EMBO Mol Med 2011; 3:623–636. 74. Guo W, Wu S, Liu J, Fang B. Identification of a small molecule with synthetic lethality for K-ras and protein kinase C iota. Cancer Res 2008; 68:7403– 7408.
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Ras in digestive oncology Charette et al. 75. Luo J, Emanuele MJ, Li D, et al. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 2009; 137:835– 848. 76. Ahearn IM, Haigis K, Bar-Sagi D, Philips MR. Regulating the regulator: posttranslational modification of RAS. Nat Rev Mol Cell Biol 2012; 13:39–51. 77. Kato K, Cox AD, Hisaka MM, et al. Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc Natl Acad Sci U S A 1992; 89:6403–6407. 78. Sinensky M, Beck LA, Leonard S, Evans R. Differential inhibitory effects of lovastatin on protein isoprenylation and sterol synthesis. J Biol Chem 1990; 265:19937–19941. 79. Lee J, Hong YS, Hong JY, et al. Effect of simvastatin plus cetuximab/irinotecan && for KRAS mutant colorectal cancer and predictive value of the RAS signature for treatment response to cetuximab. Invest New Drugs 2014. [Epub ahead of print] Shows that high-dose simvastatin may reverse cetuximab resistance in KRAS mutant colorectal cancer, with limited toxicity. 80. Van Cutsem E, van de Velde H, Karasek P, et al. Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer. J Clin Oncol 2004; 22:1430–1438. 81. Rao S, Cunningham D, de Gramont A, et al. Phase III double-blind placebocontrolled study of farnesyl transferase inhibitor R115777 in patients with refractory advanced colorectal cancer. J Clin Oncol 2004; 22:3950– 3957. 82. Marom M, Haklai R, Ben-Baruch G, et al. Selective inhibition of Ras-dependent cell growth by farnesylthiosalisylic acid. J Biol Chem 1995; 270:22263– 22270. 83. Haklai R, Weisz MG, Elad G, et al. Dislodgment and accelerated degradation of Ras. Biochemistry 1998; 37:1306–1314.
84. Laheru D, Shah P, Rajeshkumar NV, et al. Integrated preclinical and clinical development of S-trans, trans-farnesylthiosalicylic acid (FTS, Salirasib) in pancreatic cancer. Invest New Drugs 2012; 30:2391–2399. Shows that Ras inhibition is achievable in pancreas cancer and paves the way for further clinical evaluation of salirasib in pancreas adenocarcinoma. 85. Charette N, De SC, Horsmans Y, et al. Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms. Cell Death Dis 2013; 4:e471. 86. Charette N, De Saeger C, Lannoy V, et al. Salirasib inhibits the growth of hepatocarcinoma cell lines in vitro and tumor growth in vivo through ras and mTOR inhibition. Mol Cancer 2010; 9:256. 87. Halaschek-Wiener J, Wacheck V, Schlagbauer-Wadl H, et al. A novel Ras antagonist regulates both oncogenic Ras and the tumor suppressor p53 in colon cancer cells. Mol Med 2000; 6:693–704. 88. McCubrey JA, Steelman LS, Chappell WH, et al. Ras/Raf/MEK/ERK and PI3K/ && PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance. Oncotarget 2012; 3:1068–1111. An extensive review of Ras downstream signalling inhibitors. 89. Grothey A, Van CE, Sobrero A, et al. Regorafenib monotherapy for previously & treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet 2013; 381:303–312. Shows that regorafenib increase overall survival in chemorefractory metastatic colorectal cancer. 90. Samalin E, Bouche O, Thezenas S, et al. Sorafenib and irinotecan (NEXIRI) as & second- or later-line treatment for patients with metastatic colorectal cancer and KRAS-mutated tumours: a multicentre phase I/II trial. Br J Cancer 2014; 110:1148–1154. Shows that sorafenib has promising activity in chemorefractory metastatic colorectal cancer. &
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