Abstract

The pancreas is a gland composed mainly by endocrine and exocrine cells, giving rise to three main tumour types. Pancreatic neuroendocrine tumour or PNET arise from the endocrine portion of the pancreas. On the contrary, pancreatic exocrine neoplasms include pancreatic ductal adenocarcinoma (PDAC) and acinar cell carcinoma. PDAC is the most common type of pancreatic cancer and one of the leading causes of cancer-related death. It has been shown that less than 3% of PDAC patients have an overall survival of up to 5 years in the U.K. This mainly arises since the majority of patients diagnosed with PDAC present with advanced unresectable disease, which is highly resistant to all forms of chemotherapy and radiotherapy. Activating mutations of an isoform of the RAS protein, KRAS, are found in almost all PDAC cases and occur during early stages of malignant transformation. KRAS mutations play a critical role as they are involved in both initiating and maintaining PDAC development. The interaction of RAS with GDP/GTP along with its recruitment to the membrane affects transduction of its activating signals to downstream effectors. In this review, we aim to summarise different mutations of RAS and their prevalence in pancreatic cancer along with other RAS-induced tumours. In addition, we briefly discuss the genetically engineered mouse models that have been developed to study KRAS-mutated adenocarcinomas in the pancreas. These provide an opportunity to also address the importance of targeting RAS for better treatment response in PDAC patients along with the challenges incurred herein.

Introduction

Discoveries of the transforming activities of Harvey and Kirsten rat sarcoma retroviruses led to the identification of RAS genes, HRAS and KRAS, which were found to promote tumourigenesis [1,2]. The cellular homologues of the retroviruses genes were first identified in the rat genome in 1981 [3]. Ensuing this discovery, several other studies classified RAS as an oncogene in human and mouse genome [46]. Later in 1983, the NRAS isoform was identified from the cloning of neuroblastoma and leukaemia cell lines [7,8].

The three RAS isoforms (K, H and NRAS) belong to the RAS gene family, which fall under the RAS superfamily proteins. This superfamily of proteins shares the biochemical activity of GTP binding and hydrolysis and consists of four other distinct subfamilies including Rho/Rac, Rab, Arf and Ran [9,10]. Unlike other RAS isoforms, KRAS gene is subjected to alternative RNA splicing, giving rise to two splice variants, termed as KRAS4A and KRAS4B. These two transcripts are encoded by two alternative fourth exons [11,12].

The RAS family has been one of the most studied groups of proteins for the last four decades. Their prominence is due, in part, to their indispensable function in governing cell growth, proliferation, differentiation, apoptosis, tumourigenesis and tumour progression [13]. With this review, we aim to address the role of the RAS family in cancer development with a focus on KRAS in pancreatic cancer.

RAS protein activation and signal transduction

RAS proteins (∼21 kDa) function as GTPases, shuttling between the GTP-bound active and GDP-bound inactive form, regulated by guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively. Several factors, including oncogenic mutations, attenuates RAS interaction with GAPs and impairs intrinsic and GAP-mediated GTP hydrolysis. This in turn accentuates GEFs to activate RAS proteins and keeps them in the constitutively active state [1416]. Further investigations revealed that while this loss of GTP hydrolysis is important, sustained activation of the RAS protein is maintained by the recruitment of the protein to the membrane. This protein anchorage to the membrane usually takes place due to its post-translational modifications by prenylation and palmitoylation [8,17]. The membrane localisation of RAS proteins is crucial in enabling them to remain in close proximity to GEFs and consequently leading to the formation of active GTP-RAS [18]. This plays a pivotal role in regulating several downstream signalling effects as the effector proteins have a stronger binding affinity to GTP-RAS [1921].

The binding of RAS to its effectors is mainly governed by its molecular switches [10]. These switches, switch I (S-I) and switch II (S-II), are motile regions placed at the G domain of the RAS N-terminus [22]. Amino acid residues 1–86 of the G domain, which constitute an effector lobe, contain highly homologous sequences among RAS isoforms. The remaining residues 87–166 of the domain comprise an allosteric lobe that shares 82% sequence similarity [23] and is, indeed, important for enabling binding of RAS to the membrane [24,25].

Several factors regulate RAS activation and ultimately result in a plethora of downstream effects. Upstream of RAS, different external stimuli, including growth factors and mitogens, play a role in the activation and intracellular signal transduction of the RAS proteins. Receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR), upon stimulation, allow the adaptor protein growth factor receptor-bound protein-2 (GRB2) to recruit GEF to the plasma membrane, in close proximity to RAS [2629]. These GEF proteins, which include Son of Sevenless (SOS), in turn replace GDP with GTP in RAS, allowing the protein activation. In normal wild-type conditions, GAP play a role in converting RAS proteins from active into inactive state by mediating GTP hydrolysis [8], thus ensuring a termination of RAS activity (Figure 1).

Downstream effectors of RAS signalling pathway.

Figure 1.
Downstream effectors of RAS signalling pathway.

The figure summarises a plethora of pathway effectors stimulated by active RAS-GTP. GDP-bound inactive RAS is converted to active GTP-RAS through GEFs. Active RAS recruits RAF proteins to the plasma membrane and activates it. Subsequently, activated RAFs in turn lead to phosphorylation and activation of MEK and ERK proteins in a signalling cascade. PI3K is the second best-known effector of RAS-GTP. RAS-mediated activation of the catalytic subunit p110 of PI3K stimulates the kinase activity of PKB/Akt via PIP3. RAS activation in MAPK and PI3K signalling pathways regulates several cellular processes including cell cycle progression, survival, transcription and migration. Ral-GDS, also an important effector of RAS-GTP, gets activated as it gets recruited to the plasma membrane to target Ral. Ral involvement in PLD regulation results in Golgi trafficking vesicle formation. Similarly, activation of the JNK pathway by Ral regulates gene expression. RAS-GTP binds to Nore1 and Nore1/RASSF1 in turn leads to the activation of proapoptotic proteins MST1. Interestingly, cell migration and organisation of actin cytoskeleton is also regulated by RAS-GTP-mediated activation of Tiam1/Rac-GTP/PAK.

Figure 1.
Downstream effectors of RAS signalling pathway.

The figure summarises a plethora of pathway effectors stimulated by active RAS-GTP. GDP-bound inactive RAS is converted to active GTP-RAS through GEFs. Active RAS recruits RAF proteins to the plasma membrane and activates it. Subsequently, activated RAFs in turn lead to phosphorylation and activation of MEK and ERK proteins in a signalling cascade. PI3K is the second best-known effector of RAS-GTP. RAS-mediated activation of the catalytic subunit p110 of PI3K stimulates the kinase activity of PKB/Akt via PIP3. RAS activation in MAPK and PI3K signalling pathways regulates several cellular processes including cell cycle progression, survival, transcription and migration. Ral-GDS, also an important effector of RAS-GTP, gets activated as it gets recruited to the plasma membrane to target Ral. Ral involvement in PLD regulation results in Golgi trafficking vesicle formation. Similarly, activation of the JNK pathway by Ral regulates gene expression. RAS-GTP binds to Nore1 and Nore1/RASSF1 in turn leads to the activation of proapoptotic proteins MST1. Interestingly, cell migration and organisation of actin cytoskeleton is also regulated by RAS-GTP-mediated activation of Tiam1/Rac-GTP/PAK.

The first bona fide downstream effector of RAS protein, discovered by four groups in 1993, was named RAF1 [3033]. Since then, extensive research led to the elucidation of mitogen-activated protein kinase (MAPK) signalling pathways. Briefly, RAF1 phosphorylates subsequent downstream target, the mitogen-activated protein kinase kinase (MEK). MEK, as a dual-specificity kinase, phosphorylates both serine/threonine and tyrosine sites of the downstream extracellular signal-regulated kinase (ERK) 1/2 [8]. The RAS/RAF/MEK/ERK pathway is involved in various biological processes including cell cycle regulation, apoptosis, proliferation, migration and differentiation [34]. Several other effector proteins have been shown to be regulated downstream of RAS, including Ral guanine nucleotide dissociation stimulator (Ral-GDS), phosphatidylinositol 3-kinase (PI3K), AF6, p120GAP, RASSF/NORE1, Tiam, Rin1, PLCε and PLCζ [18]. Interestingly, an anti-apoptotic role for RAS-GDP has been shown to occur through the Aiolos transcription factor which binds RAS-GDP and in turn directly modulates the expression of Bcl-2 [35]. The RASSF family are RAS effectors that restrict pathway activation by disrupting RAS interaction with RAF1 [36] and by promoting several pathways, such as Rb activation [37], the hippo pathway proapoptotic signal [38] and p53 signalling in response to KRAS activation [39]. Interestingly, loss of RASSF family members in the endocrine pancreas has been reported in association with pathologies related to islet or acinar cells [4044].

Due to the high degree of sequence homology, functional specificity of each of the RAS isoforms was originally ruled out. However, several studies have now shown distinct functions of each RAS isoform, as they associate with particular signalling networks and lead to different functional output and cellular phenotype. For example, KRAS has higher potency in activating RAF-1 in comparison with HRAS, whereas HRAS displays greater ability to activate PI3K [45]. In many cancers, a specific RAS isoform, mostly KRAS, has greater prevalence [10]. Interestingly, studies using a mouse model with a knockout of both Kras alleles were shown to be embryonically lethal. Heart abnormalities were observed in Kras−/− mice; embryos developed normally in mice that were deficient for other RAS isoforms, Hras and Nras. This indicated Kras to be the only necessary isoform during mouse development [46,47].

RAS mutations in the cancer landscape

RAS genes and isoforms were identified as oncogenes with neoplastic potential more than three decades ago [4,4850]. It is mutationally activated in almost 33% of all human cancers, making it one of the most common oncogenic mutations. The most mutated isoform is KRAS, followed by NRAS and HRAS [51,52]. Single base substitution in codons 12, 13 and 61 (G12, G13 and Q61) in RAS oncogenes are the most common mutations, resulting in a constitutive activation of RAS proteins [25]. KRAS is predominantly mutated at G12 (83% of all mutations), whereas NRAS and HRAS show variable prevalence for all the three mutation sites. Replacement at codons 12 or 13 by any amino acid except proline creates a steric block that inhibits the hydrolysis of RAS-GTP to RAS-GDP [53]. The aberrant up-regulation of RAS and its downstream effectors results in many of the phenotypic hallmarks of cancer including increased proliferation, suppression of apoptosis, altered metabolism, change in the tumour microenvironment, evasion of the immune response and metastasis [8,25,51,54,55]. In Figure 2, we summarise the mutation prevalence and tumour correlation for the RAS isoforms.

RAS mutations in human cancer.

Figure 2.
RAS mutations in human cancer.

Summary of mutations of all RAS isoforms in different types of human cancer. (A), Genomic position of the three RAS isoforms KRAS, NRAS and HRAS in the human chromosome and relative pie charts with the percentage of mutation in the three main codons, G12, G13 and Q61. (B) Diagram of the human body with different tumours and relative percentages represent frequency of mutations for each RAS isoform [22,69], the outer pie chart refers to the frequency of mutation in the RAS isoforms, the inner pie chart shows the frequency of mutated codons within KRAS isoform.

Figure 2.
RAS mutations in human cancer.

Summary of mutations of all RAS isoforms in different types of human cancer. (A), Genomic position of the three RAS isoforms KRAS, NRAS and HRAS in the human chromosome and relative pie charts with the percentage of mutation in the three main codons, G12, G13 and Q61. (B) Diagram of the human body with different tumours and relative percentages represent frequency of mutations for each RAS isoform [22,69], the outer pie chart refers to the frequency of mutation in the RAS isoforms, the inner pie chart shows the frequency of mutated codons within KRAS isoform.

KRAS is located in the short arm (p) of the human chromosome 12 (12p12.1-pter). Codons 12 and 13 are the most common mutational sites. KRAS mutations are most common in PDAC, colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) with codon 12 being the mutational site in nearly 90% of cases in PDAC [56]. Extensive research has provided a detailed genetic profile of PDAC with KRAS mutation found in ∼95% of patients [5659]. It is an early event in PDAC progression with over 90% of low-grade pancreatic intraepithelial neoplasia (PanIN) lesions harbouring oncogenic KRAS mutations [60]. PanINs are graded from stage I to III, and along this continuum, they display increasing disorganisation and nuclear abnormalities, with high-grade PanINs ultimately transforming into aggressive PDAC. The prognostic value of KRAS mutation and the role of different alleles have been examined in several studies. In PDAC, the G12D mutation is associated with a lower overall survival whereas patients with G12R have a far better clinical outcome [61].

In the endocrine cells of the pancreas, that include cells of the islets of Langerhans, activated KRASG12D rather play a paradoxical role by displaying anti-proliferative effect. This occurs due to the presence of the tumour suppressor gene menin, MEN1, which inhibits the MAPK pathway signalling and instead favours RASSF1A-mediated anti-proliferative effect, downstream of RAS [40].

Genetically engineered mouse models for RAS-induced tumours

Brinster, Paltimer and co-workers in early 1987, undertook the first step towards the understanding of RAS signalling in cancer development. They reported that ectopic expression of a human HRAS oncogene in acinar cells under the control of the Elastase promoter induced massive damage in the foetal pancreas [13,62]. Later, gene knockout studies have revealed profound differences among the RAS isoforms. Hras or Nras knockout mice, and even double knockout mice, are viable and show no obvious phenotypic abnormalities [63,64], whereas knockout of the Kras gene is embryonically lethal. In 2001 Jackson and colleagues developed a Kras+/LSLG12D mouse model, later described as the ‘gold standard’ for most Kras oncogene-driven animal tumour models. The inducible expression of mutant Kras alleles promotes the development of preneoplastic epithelial hyperplasia, adenoma and adenocarcinoma in the lung and gastrointestinal tract. Similarly, in the pancreas conditional Kras expression promotes PanIN and PDAC. Only low frequencies of these Kras-driven lesions progress to invasive and metastatic cancer.

More invasive and aggressive phenotypes are developed predominantly in the presence of other tumour suppressor disabling mutations, such as in Tp53 [65], Smad4 [66], Pten [67] and activation of Wnt/beta-catenin signalling [68]. A striking example of the different phenotype of genetically engineered mouse models (GEMMs) carrying Kras mutation alone or in concomitance with other mutated gene is given by the KrasG12D; Pdx1-cre (KC) model and the LSL-KrasG12D; LSL-Trp53R172H; Pdx1-cre (KPC) model reported by Hingorani and colleagues in 2003 and 2005, respectively [69,70]. Through the Cre-Lox technology, the expression of mutant endogenous alleles in the KC and KPC model is regulated by the pancreas-specific Pdx1 promoter. Pdx1 is fundamental for the development of the pancreas, Pdx1+ epithelial cells will eventually give rise to the whole pancreas — its exocrine, endocrine and ductal cell populations [71]. Pdx1 expression first starts at mouse embryonic day 8.5–9.0 (E8.5–9.0) and continues until E12.0–E12.5 [72]. The KPC model develops the complete spectrum of pre-invasive PanIN, as well as end-stage pancreatic cancer and exhibits the clinical features and similar aggressive metastatic phenotype with 100% penetrance and with a much shorter latency relative to the KC model.

Barbacid and colleagues reported a second generation of GEM tumour models. Their model is generated by crossing mice carrying a knocked-in KrasLSLG12Vgeo allele with double transgenic mice (Elas-tTA; Tet-O-Cre) that express Cre recombinase under the control of the Elastase promoter, following an inducible Tet-Off strategy [73,74]. Differently from KC and KPC model, here the Cre recombinase is controlled by the pancreatic elastase promoter, expressed in the acinar cells only. Using this Tet-Off strategy, the KrasG12V oncogene expression can be controlled in a temporal manner by providing doxycycline in drinking water. Untreated mice express the resident KrasG12V in a limited percentage of acinar cells (20–30%) during late embryonic development [74]. The mice develop PanIN lesion resembling the latencies and penetrance of the models expressing KrasG12D. As for the models expressing KrasG12D, in the presence of other inactivated tumour suppressor genes, the penetrance of tumour development increases at 100% with shortened latency [73]. In Table 1, the standard of the field of the Kras mice models is summarised.

Table 1
Overview of the genetically engineered mice models for KRAS mutation and their phenotypes
Mouse model Phenotype References 
KrasG12D; Pdx1-Cre KC model, full spectrum of pre-invasive PanIN, progressing to invasive and metastatic PDAC at low frequency Hingorani et al. [69
KrasG12D; Ink4a/Arflox/lo; Pdx1-Cre KIC model, Development of aggressive tumours and replicates several clinical features of PDAC; rarely present metastasis Aguirre et al. [75]; Bardeesy et al. [65
KrasG12D; Trp53mutant; Pdx1-Cre KPC model, pancreatic cancer displaying all the associated features of the human disease with similar aggressive and metastatic phenotype Hingorani et al. [70
KrasG12D; TGF-βR2flox; Ptf1/p48-Cre Has normal pancreatic development with the rapid growth of well-differentiated pancreatic cancer with associated weight loss, haemorrhagic ascites and jaundice; develops metastasis predominately in the liver Ijichi et al. [76
K-RasLSLG12Vgeo; Elastase-tTA; Tet-O-Cre; Using a tet-off strategy to express Cre, KRASG12V expression can be temporally regulated. Expression of KRASG12V in pancreatic precursors during early embryonic development induces the progressive development of PanINs histologically indistinguishable from those observed in patients When KRASG12V expression is restricted to adult acinar cells, only upon treatment with caerulein, 100% of the mice develop PanINs Guerra et al. [74
KrasLSLG12D/+,Dpc4flox/flox; Ptf1/p48-Cre KDD model, develop aggressive, local invasive and widely metastatic pancreatic tumours Izeradjene et al. [77
KrasG12D; Ptenflox; Pdx1-Cre Rapid progression of PanIN and tumour development; local invasive tumour with rare metastasis Hill et al. [67]; Kennedy et al. [78
KrasG12D; Trp53flox/flox; Cre or KrasG12D; Trp53mut/mut; Pdx1-Cre KPPC model, rapid tumour development; reduced median survival compared with mice carrying heterozygous deletion or mutation of Trp53; multifocal tumours, jaundice, ascites and local invasion occur; metastasis is not a common feature Morton et al. [79
 Kras+/LSLG12Vgeo; Elastase-tTA; p53lox/lox; Tet-O-Cre Using a tet-off strategy to express Cre, KRASG12V expression can be temporally regulated. Expression of KRASG12V in pancreatic precursors during early embryonic development induces the progressive development of PanINs histologically indistinguishable from those observed in patients When KRASG12V expression is restricted to adult acinar cells, only upon treatment with caerulein, 100% of the mice develop PanIN Guerra et al. [80
Kras+/LSLG12Vgeo; Elastase-tTA; Ink4a/Arflox/lox; Tet-O-Cre The temporally regulated (tet-off strategy) expression of K-Ras oncogenes and loss of p16Ink4a/p19Arf in embryonic acinar cells frequently result in aggressive tumour as anaplastic carcinomas, seldom observed when these mutations are induced in the adult animal Guerra et al. [80
KrasG12D; Trp53R172H/+; Ink4flox/+; Ptf1/p48-Cre KPIC model, mice develop adenocarcinoma that closely resembles human PDAC, characterize by rich desmoplastic stroma and low microvascularity Ma and Saiyin [81
Mouse model Phenotype References 
KrasG12D; Pdx1-Cre KC model, full spectrum of pre-invasive PanIN, progressing to invasive and metastatic PDAC at low frequency Hingorani et al. [69
KrasG12D; Ink4a/Arflox/lo; Pdx1-Cre KIC model, Development of aggressive tumours and replicates several clinical features of PDAC; rarely present metastasis Aguirre et al. [75]; Bardeesy et al. [65
KrasG12D; Trp53mutant; Pdx1-Cre KPC model, pancreatic cancer displaying all the associated features of the human disease with similar aggressive and metastatic phenotype Hingorani et al. [70
KrasG12D; TGF-βR2flox; Ptf1/p48-Cre Has normal pancreatic development with the rapid growth of well-differentiated pancreatic cancer with associated weight loss, haemorrhagic ascites and jaundice; develops metastasis predominately in the liver Ijichi et al. [76
K-RasLSLG12Vgeo; Elastase-tTA; Tet-O-Cre; Using a tet-off strategy to express Cre, KRASG12V expression can be temporally regulated. Expression of KRASG12V in pancreatic precursors during early embryonic development induces the progressive development of PanINs histologically indistinguishable from those observed in patients When KRASG12V expression is restricted to adult acinar cells, only upon treatment with caerulein, 100% of the mice develop PanINs Guerra et al. [74
KrasLSLG12D/+,Dpc4flox/flox; Ptf1/p48-Cre KDD model, develop aggressive, local invasive and widely metastatic pancreatic tumours Izeradjene et al. [77
KrasG12D; Ptenflox; Pdx1-Cre Rapid progression of PanIN and tumour development; local invasive tumour with rare metastasis Hill et al. [67]; Kennedy et al. [78
KrasG12D; Trp53flox/flox; Cre or KrasG12D; Trp53mut/mut; Pdx1-Cre KPPC model, rapid tumour development; reduced median survival compared with mice carrying heterozygous deletion or mutation of Trp53; multifocal tumours, jaundice, ascites and local invasion occur; metastasis is not a common feature Morton et al. [79
 Kras+/LSLG12Vgeo; Elastase-tTA; p53lox/lox; Tet-O-Cre Using a tet-off strategy to express Cre, KRASG12V expression can be temporally regulated. Expression of KRASG12V in pancreatic precursors during early embryonic development induces the progressive development of PanINs histologically indistinguishable from those observed in patients When KRASG12V expression is restricted to adult acinar cells, only upon treatment with caerulein, 100% of the mice develop PanIN Guerra et al. [80
Kras+/LSLG12Vgeo; Elastase-tTA; Ink4a/Arflox/lox; Tet-O-Cre The temporally regulated (tet-off strategy) expression of K-Ras oncogenes and loss of p16Ink4a/p19Arf in embryonic acinar cells frequently result in aggressive tumour as anaplastic carcinomas, seldom observed when these mutations are induced in the adult animal Guerra et al. [80
KrasG12D; Trp53R172H/+; Ink4flox/+; Ptf1/p48-Cre KPIC model, mice develop adenocarcinoma that closely resembles human PDAC, characterize by rich desmoplastic stroma and low microvascularity Ma and Saiyin [81

Treatment and challenges

Despite several efforts to target KRAS for therapy, the attempts made so far have been unsuccessful. This has even led to label KRAS as being the ‘undruggable’ target. This title has indeed been inferred by certain biochemical and structural properties of the protein. In particular, the biochemical function of GTP hydrolysis, enabled by the intrinsic GTPase activity of the protein along with the support of GAP, allows KRAS to transit from GTP-bound active to GDP-bound inactive state, as discussed earlier. However, mutation of KRAS proteins makes them more favourable for GTP-bound active states. Previous efforts were made to tackle these mutated active KRAS proteins through the discovery of GTP-competitive inhibitors. However, such attempts proved ineffective due to high-affinity GTP interaction with KRAS in the picomolar range [82]. Structurally, the lack of deep and large hydrophobic binding site at the surface of KRAS catalytic domain made the proteins incompatible for targeting by high-affinity small binding molecules [8386].

Bournet and colleagues in 2009 presented KRAS mutation as a reliable biomarker for PDAC. This was suggested from their study that combined KRAS mutation assay with cytopathological examinations from endoscopic ultrasound (EUS)-guided fine needle biopsy (FNS) in patient samples [87,88]. Similarly, detection of wild-type KRAS was associated with improved patient survival [21]. Strikingly, the ratios of wild-type and mutant alleles in KRAS played an important role in PDAC prognosis [89]. This was further reinforced from the study using the dataset developed by Biankin and colleagues where the higher allelic ratio was in correlation with shorter overall survival [56]. Interestingly, Mueller and colleagues, using mouse model with conditional KrasG12D in the pancreas, also suggested that the increase in KrasG12D gene dosage due to allelic imbalances played a role in the early progression of PDAC and tumour metastasis [90]. Owing to the very high percentage of PDAC cases with heterogeneity in KRAS-mutated alleles, it would be useful to tailor the investigation based on allelic ratio and mutated KRAS dosage specific to each tumour [91]. Despite these potential roles of KRAS in both diagnosis and prognosis, KRAS mutational status has yet to predict therapeutic response in PDAC patients [21].

Inhibitors, including lonafarnib and tipifarnib, which target farnesyl transferase, were tested in Phase III clinical trials. The enzyme farnesyl transferase is involved in the membrane recruitment of RAS through the process of prenylation [19]. However, the inhibitors were unsuccessful in improving the clinical outcome of PDAC patients [92]. This occurs possibly due to maintained prenylation by an alternative enzyme, geranylgeranyltransferase I, leading to the sustained anchorage of the RAS to the membrane [21]. An inhibitor with dual specificity for farnesyl transferase and geranygeranyltransferase I, L-778 123 was tested in Phase I clinical trial in combination with radiotherapy in locally advanced pancreatic cancer patients [93]. However, concerns related to cardiac safety led to the termination of the trial [93,94].

Furthermore, downstream effectors of RAS, including MEK in MAPK signalling, have been targeted to treat tumours with RAS mutations. Small molecule MEK inhibitors, selumetinib and trametinib, have been tested in phase I and II studies, either alone or in combination with gemcitabine, which is one of the main chemotherapy drugs to treat patients with PDAC. However, issues such as toxicity and acquired resistance diluted their effectiveness in abrogating RAS-induced downstream signals [21].

Interestingly, antisense technology has been developed and successfully tested to specifically target point-mutated KRAS in pancreatic cell lines [95,96]. The antisense oligonucleotide ISIS-2503 inhibits the expression of human HRAS mRNA and was tested in patients with locally advanced or metastatic PDAC in Phase II trial in combination with gemcitabine. The primary outcome measure in this study included overall survival of more than 6 months in 58% of patients, which was in line with their predefined criteria for success. However, the benefits of this combination treatment were not well-defined in patients suffering from metastatic disease [97]. The treatment selection was also limited as it did not include PDAC patients with non-resectable tumour. Similarly, it was undefined whether the treated patients had decreased expression of HRAS, which was identified as the valid target from the in vitro study.

Khvalevsky and colleagues demonstrated direct targeting of KRAS by RNA interference approach for the treatment of pancreatic cancer. By encapsulating siRNA against mutated KRASG12D in a biodegradable polymer matrix, they showed a reduction in tumour growth in mice along with an increase in their survival rate. Additionally, they recapitulated these effects in vitro by demonstrating changes in the cellular phenotype including inhibition of proliferation and epithelial-to-mesenchymal transition (EMT). This could provide an effective therapeutic approach since the siRNA is prevented from degradation and is provided locally to solid pancreatic tumours in a controlled manner for prolonged periods [98].

Perspectives
  • Importance of the field: KRAS is the most frequently mutated isoform in PDAC and is quoted to be associated with 90% of pancreatic cancers. Hence, the primary focus entailed either directly blocking the activation of KRAS protein or perturbing signalling pathways contributing to KRAS activity. Interestingly, the National Cancer Institute also listed KRAS targetting as one of the critical components in the field of pancreatic cancer research.

  • Current thinking: KRAS mutations in PDAC have been hypothesised to promote tumour initiation and evade apoptosis and senescence, allowing promotion of cancer. Several attempts have also been made in the past to disrupt the binding of RAS to its effectors. These include hindering active conformations of the motile S-I and S-II regions and affecting the nucleotide binding site [99102]. Inhibitors that covalently bind to the P loop region of switch II (S-IIP) prevent the GDP-bound KRAS from converting the protein to its active conformational state. 1–4% of pancreatic cancers have KRASG12C mutation [103,104]. S-IIP inhibitors, including ARS-853 and its analogues, were shown to reduce KRAS-GTP in cells with KRASG12C mutation via covalent occupancy of Cys-12 by the inhibitors. However, these failed to show any effect in vivo. Janes and co-workers demonstrated that the compound ARS-1620 was able to achieve covalent target occupancy of GDP-bound KRASG12C using an in vivo model as well as G12C KRAS-mutated cell lines. Active KRAS-GTP was inhibited, leading to tumour regression in a patient-derived tumour xenograft model [105]. These approaches are specific for 12C, not a mutation commonly seen in PDAC, and it is not clear that such targeting of 12 V/D/R also can be accomplished with similar results. Despite these discoveries, many challenges have been encountered during direct targeting of the RAS proteins. Hence, more effective strategies would still require focussing on disrupting downstream effector signalling, including RALGEF-RAL Small GTPase, PI3K–AKT–mTOR and RAF–MEK–ERK pathways [106].

  • Future directions: As an alternative approach, immunotherapy appears to be a more viable avenue for targeting tumours with RAS mutation. KRASG12D mutation, which is found in 45% of pancreatic cancers [85], has not yet been effectively targeted by any drug. A report in 2016, based on a patient with metastatic CRC, described an anti-tumour immunotherapy-based approach. Using this approach, tumour-infiltrating lymphocytes containing cytotoxic T cells, which specifically targeted KRAS G12D, impaired tumour progression. This immune-based therapy could be beneficial in treating pancreatic cancer patients with a high frequency of the mutation [85]. Indeed, in an immunotherapy-based approach, synthetic RAS peptides, expressing residues 5–21 of the RAS proteins, were utilised for vaccination in Phase I/II trial in pancreatic cancer patients with resected tumours [107]. More recently, Phase I/II clinical trial in the Targovax study has applied the adjuvant vaccination, TG01, in pancreatic cancer patients, along with gemcitabine as standard chemotherapy treatment. Patient cohorts in this study displayed a strong 2-year survival rate [108]. This provides an exciting platform that can be explored further. Importantly, it also demands the need to develop therapies targeting RAS mutations in order to improve PDAC patient survival.

Abbreviations

     
  • CRC

    colorectal cancer

  •  
  • ERK

    extracellular signal-regulated kinase

  •  
  • GAP

    GTPase-activating proteins

  •  
  • GEF

    guanine nucleotide-exchange factor

  •  
  • GEMMs

    genetically engineered mouse models

  •  
  • KPC

    KrasG12D; LSL-Trp53R172H; Pdx1-cre

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MEK

    mitogen-activated protein kinase kinase

  •  
  • PanIN

    pancreatic intraepithelial neoplasia

  •  
  • PDAC (30)

    pancreatic ductal adenocarcinoma

  •  
  • PI3K

    phosphatidylinositol 3-kinase

  •  
  • Ral-GDS

    Ral guanine nucleotide dissociation stimulator

  •  
  • S-II

    switch II

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contributed equally to this work.