RAS-mediated oncogenic signaling pathways in human malignancies

Abdul Q. Khan, Shilpa Kuttikrishnan, Kodappully S.Siveen, Kirti S. Prabhu, Muralitharan Shanmugakonar, HamdaAl Naemi, Mohammad Haris, Said Dermime, Shahab Uddin

Abstract

RAS-mediated oncogenic signaling pathways in human malignancies pathological conditions including cancer. In general, RAS is the main upstream regulator of the highly conserved signaling mechanisms associated with a plethora of important cellular activities vital for normal homeostasis. Mutated or the oncogenic RAS aberrantly activates a web of interconnected signaling pathways including mitogenactivated protein kinases (MAPK), phosphoinositide-3 kinase (PI3K)/AKT pathways, protein kinase C (PKC) and ral guanine nucleotide dissociation stimulator (RalGDS), etc., leading to uncontrolled transcriptional expression and reprogramming in the functioning of a range of nuclear and cytosolic effectors critically associated with the hallmarks of carcinogenesis. This review highlights the recent literature on how oncogenic RAS negatively use its signaling web in deregulating the expression and functioning of various effector molecules in the pathogenesis of human malignancies.

Key Words: RAS proteins, Oncogenes, Mutation, Signaling, Malignancies,

1. Introduction

Cancer is one of the leading causes of morbidity and mortality throughout the globe. Over the years it has been discovered that a number of genes/proteins play critical role in carcinogenesis. Irreversible changes in the cellular genetic content are considered to be the major cause of carcinogenesis as it can modulate both gene expression and functioning of translational product (proteins) involve in regulating cell growth and differentiation. With the advent of modern technologies and continuous efforts of researchers enormous progress has been made in the identification and understanding of the role of gene mutations/alterations in array of human malignancies and in carcinogenesis. In this respect, a gene family which has always been found to be mutated in most of the human cancers and also the leading focus of research in understanding the process of carcinogenesis and drug development is the RAS.
RAS proteins belongs to small protein GTPase family with intrinsic catalytic (GTPase) activity and are considered as the major regulator of a plethora of molecular and cellular events including cellular growth and proliferation, cytoskeleton integrity, cell adhesion and migration, differentiation, and survival in the biological system ranging from yeast to humans. Significant amount of work has been done and been published on RAS signaling machinery in human health and normal homeostasis.

2. Structure of RAS

RAS is the first recognized protein category known to regulate cell proliferation and growth in retroviruses. So far an array of RAS proteins were identified and categorized in five major families namely; Ran, Ras, Rab, Rho, and Arf [1]. Structurally, RAS family of proteins are most extensively studied and are further grouped as Di-Ras, Rap, Ral, RheB/RheB-like, Rerg etc., in extended RAS family [2]. RAS are the guanosinenucleotide-binding proteins which consists of around equal number of amino acids and polypeptides with 21 KDa of mass having strands and helices (α-helices, β-strands) with two domains (G and C). G domain binds with guanosine-nucleotide and attaches with a hypervariable C-terminal domain which helps in membrane attachment [3]. Hypervariable C-terminal domain terminates with a CAAX motif, where cysteine is prenylated by farnesyltransferase (FTase). The –AAX portion left after prenylation is removed by RAS-converting enzyme 1. Isoprenylcystein-carboxyl-methyltransferase (ICMT) then methylates carboxyl group of the newly exposed isoprenylcysteine. Lipid modifications formed by this reaction provide weak membrane binding affinity which is stabilized by a second signal motif. These modifications help in localization of these isoforms at cellular levels [4].
The peculiar structural similarities in members of RAS family proteins are evolutionally conserved from lower animal species like yeast to humans. This supports the ubiquitous nature of RAS proteins and suggests the importance of RAS proteins in growth and development from lowest to highest eukaryotes species. RAS being the central master regulator/activator in major signaling pathways, has a very tightly controlled “on” (RASGTP) and “off” (RAS-GDP) action efficiently executed by guanine nucleotide exchange factors (GNEFs) and GTPase activating proteins (GAPs) that facilitate conversion of GDP to GTP and vice versa [5, 6]. An array of stimuli such as receptor tyrosine kinases, non-receptor tyrosine kinase-associated receptors, G protein-coupled receptors and integrins etc., are known to promote RAS activation [6, 7].

3. RAS and carcinogenesis

Carcinogenesis is a complex and multistep process acquaint by accretion of the number of genetic and epigenetic changes in the expression and functioning of oncogenes, tumor-suppressor genes, and microRNA genes [8]. Harvey rat sarcoma viral oncogene homolog (H-RAS), Kirsten rat sarcoma viral oncogene homolog (K-RAS), and neuroblastoma RAS viral (v-ras) oncogene homolog (N-RAS) were first identified as genes with oncogenic potential and since then huge number of research investigation summarizes that around 30 % of all human cancers revealed the RAS mutation as one of the prime reasons for carcinogenesis [9]. Surprisingly about half of the human colon malignancies and around 90% cases of all pancreatic cancer are detected with oncogenic mutation in RAS [10]. These conclusions suggest that mutated RAS oncogene is one of the leading causes of carcinogenesis. The oncogenic potential of RAS family of proteins such as H-RAS, K-RAS and N-RAS in various human cancers was established decades ago [11, 12]. Genomic sequencing analysis of human cancer specimen revealed that alteration in K-RAS gene is most frequent followed by N-RAS and H-RAS [12, 13]. Oncogenic alteration in K-RAS gene is most frequent in pancreatic carcinomas, colorectal tumors, and lung malignancies while mutated H-RAS is most frequent in dermatological and head and neck cancer malignancies, however hematological malignancies are often detected with N-RAS mutations [12]. Most of the mutations in RAS oncogenes are single base substitutions in 12, 13 and or 61 codons which lead to the constitutive activation of RAS proteins by inhibiting the conversion of RAS-GTP to RAS-GDP [14]. Mutated RAS oncogene triggers translation of onco-
proteins with transforming potential leading to the incessant activation of major signaling pathways associated with the cancer hallmarks [8, 14]. RAS mutation by xenobiotics exposure in the environment is also a major driving force for cancer development. Various investigation have revealed that RAS mutation is the prime cause of carcinogenesis in the animals exposed with chemical mutagens [14]. Mutagenic RAS activation is not only prime cause of carcinogenesis but is also a major driving force for making cancer cells insensitive towards therapeutic measures [11, 15-18]. Recently, it was observed that mutagenic RAS oncogene has critical role in tumor immuneresistance [19]. Indeed, mutated oncogenic RAS induced molecular and cellular alterations in carcinogenesis have proven the tumorigenic potential of mutated RAS [20, 21].

4. Oncogenic RAS mutation

RAS proteins are the translational product of three universally expressed protooncogenes namely H-RAS, K-RAS and N-RAS. Missense gain-of-function mutations are the main cause for the activation of these proto-oncogenes into oncogenes and incessant RAS activation which was revealed decades ago. Point mutations at codon 12, 13 and 61 are the most prime sites of mutation in RAS genes as shown in table 1. Functionally, these molecular alterations in RAS gene suppress the intrinsic GTPase activity that consequently leads to aberrant upregulation of RAS and its downstream effectors [12, 22-24].
H-RAS gene is present at position 5 on short arm (p) of chromosome 11 and is 6.5kb of human genome. It comprises of 6 exons and 2 alternative splice variants [25]. p21 HRAS and its negative form p 19-H-RAS are generated on splicing of exon 2 and 5 whereas, later is produced on splicing at exon 4 and exon 5 [26]. Various reports depicts that codon 12, 13 and 61 are considered to be most common spot for mutation in H-RAS gene [27-30]. K-RAS gene is located on the short arm (p) of chromosome 12 at 12.1 and consists of two splicing variants K-RAS 4A and K-RAS 4B along with 6 exons. All coding regions present in m-RNA transcripts are covered by K-RAS 4A whereas K-RAS 4B excludes exon 6 [31, 32]. Activating point mutations in K-RAS oncogene are one of the most common types of all the mutations associated with human cancers. Codons 12 and 13 are the most common mutational sites for K-RAS while codon 61 and other positions are the less frequent mutational sites [28, 33-36]. Mutations in the genetic sequence of N-RAS, another important member of RAS gene

5.1. Pancreatic carcinoma

Pancreatic adenocarcinoma is considered to be aggressive form of cancer and mostly affecting duct epithelial cells [42]. K-RAS mutation is the most frequent among the types of mutation detected at both early and chronic stage pancreatic cancer [43]. Genetic studies revealed that approximately 90% of the pancreatic cancer patients carry K-RAS mutation (table 2) [28, 44-48].In pancreatic adenocarcinoma, mutation is reported to occur at codon 12 wherein glycine is substituted with aspartic acid or with arginine or valine [49]. Several contradictory reports have been published on the extent of aggressiveness of this cancer. For instance K-RAS mutation occurring at G12R and G12A was thought to be the most aggressive form of cancer with low survival rate than compared to G12V or G12S mutation [50, 51]. Other studies have shown that GaC mutation occurring at codon 12 was found to be more aggressive than tumor bearing GaT mutations [51]. There is no effective anti-RAS therapeutic remedy developed yet for this type of cancer but agents like farsenyltransferase inhibitors acts by inhibiting post-translational modification have shown promising results in cell line and mouse xenograft models. However its use was limited as it inhibited only post translational farnesylation and not geranylation [52]. Anti-RAS agents like use of vaccination against K-RAS oncogenic peptide prolongs survival rate in surgically operated pancreatic patients gain considerable importance in treating these patients [53].

5.2. Colorectal cancer

More than 40% of cases in human colorectal cancer (CRC) are reported to harbor KRAS mutation at codons 12, 13, and 61 indicating that this mutation plays a critical role in development of tumor and associated morbidity and mortality [54-60]. Substitution of glycine (G)-to-aspartic acid (D) in codon 12 is most common in K-RAS associated CRC observed at early and late stage [54]. Low frequency of N-RAS (about 5 %) and H-RAS (about 2 %) genes mutations are reported in colorectal adenocarcinoma [61, 62]. A strong correlation is reported between poor prognosis and K-RAS mutation in CRC [63, 64]. K-RAS mutation is considered to be bad prognostic marker for patients with colorectal carcinoma having liver metastasis [65]. Similarly colorectal carcinoma patients with lung metastasis are reported to have high rate of K-RAS mutation [66]. Yet there is no specific remedy developed and use of therapeutic agents especially monoclonal antibodies against EGFR was found to be effective [67-69].

5.3. Thyroid carcinomas

Thyroid cancer is one of the most commonly diagnosed endocrine malignancies often harboring RAS mutations. Presence of all three mutant isoforms of the RAS oncogene in thyroid malignancies is unique with the predominance of N-RAS mutation in cordon 61[70]. Further, mutated RAS accounts for the second most common mutation detected in biopsies from thyroid nodules which often (80%) results into malignancy [71]. Various published reports shows difference in predicting the overall frequency of RAS mutation in thyroid carcinoma. For instance, 48% of follicular adenomas, 57% of follicular thyroid cancer, and 21% of well-differentiated papillary thyroid cancer showed mutation in RAS gene as reviewed earlier [70, 72]. Recent genetic and molecular studies revealed varying frequency of mutation in RAS oncogenes detected in different types of thyroid cancer [42, 73-75] as shown in table 2. Beside RAS, B-homologue of the rapidly accelerated fibrosarcoma (BRAF) and papillary thyroid carcinoma (RET/PTC) oncogenes mutations are also detected in thyroid carcinoma [76]. Although radioiodine is considered to be first line of treatment the adverse effects following treatment has restricted its use [77]. Clinical studies targeting BRAF mutation, RAS proteins has been found to have high success with progression free survival in thyroid cancer patients [78].

5.4. Hematopoietic malignancies

RAS genes mutation are reported to be as low as 5% in chronic myeloid leukemia and as high as 70% in chronic myelomonocytic leukemia and plasma cell myeloma [79]. KRAS and N-RAS mutation are most commonly reported forms of mutations whereas; HRAS forms are very rare. Mutation of N-RAS usually occurs at codon 61 with amino acid substitution occurring at 61Q, G12, and G13 and for K-RAS, substitution occurs at G12, G13 and Q61 [49]. About 20% of N-RAS mutation are exhibited in juvenile myelomonocytic myeloid leukemia (JMML) or plasma cell myeloma [42], while in case of chronic myelomonocytic leukemia it was detected in the range of 17-60% [42, 80]. Interestingly, mutation frequency of both K-RAS and N-RAS was detected in the range of 4-10% in patients with acute myelogenous leukemia[42, 81] and genetic molecular analysis of acute lymphoblastic leukemia samples reveals 10-15% mitogenic N-RAS and K-RAS presence [42, 82]. As mentioned under pancreatic carcinoma prognosis of RAS associated hematological malignancy remains unclear. Some study reports involvement of N-RAS in acute myelogenous leukemia to poor prognosis whereas, some published data does not correlate N-RAS with this leukemia [83-85]. Similarly in case of acute lymphoblastic leukemia N-RAS mutation is reported to have poor survival rate than compared to those bearing WT copies [86]. N-RAS mutations is correlated with weak prognostic in myelodysplastic syndrome, with a higher risk of developing acute myelogenous leukemia [87]. There is no effective therapy discovered for RAS linked hematological malignancies however, farnesyl transferase inhibitors are been used in the treatment of leukemias and lymphomas [87]. Farnesyl transferase inhibitors alone or in combination with other therapeutic agents targeting signaling pathways are under clinical trials for the treatment of leukemias and lymphomas [88].

5.5. Lung cancer

Lung cancer, one of the major concerns for cancer related death in humans, is often associated with the deregulated RAS functioning. Mutation in K-RAS is more prone than N-RAS and H-RAS occurring predominately at codon 12 followed by 13 and 61 leading to G/T transversions and often associated with poor prognosis and therapeutic outcome [89, 90]. Lung adenocarcinoma, most common histological subtype of non-small cell lung cancer (NSCLC), commonly carry K-RAS mutation with 20%-50% frequency [9193], followed by squamous cell carcinoma, subtype of NSCLC. In addition, K-RAS mutation is the most commonly observed oncogenic deriver detected in in the lung cancer patients of non-Asian origin [89].

5.6. RAS mutation in other human cancer

Despite the critical role of the oncogenic RAS signaling association of RAS mutations in other types of human cancer such as liver, breast, kidney, gynecological, and head neck cancer is low as shown in Table 2. Inline of that, presence of RAS mutation in the hepatocellular carcinoma (HCC) is comparatively low with more active in K-RAS and NRAS than H-RAS. About 7% of human liver cancers have been detected with K-RAS mutation while H-RAS and N-RAS mutations frequency is even lower [94, 95]. Recently, two separate studies showed that around 5-20% of HCC patients were detected with KRas mutation while mutations in other RAS isoforms were absent [94, 96]. In case of breast cancer, most common cancer in women, over all prevalence of RAS mutation is between 7-12%. However, variation in the frequency of K-RAS mutation often detected in breast cancer ranging from 0.9 to 20 % [97-100] while the mutation in N-RAS is least frequent [98]. Aberrant activated of the RAS associated signaling cascade is often associated with ovarian low grade serous carcinoma and its precursor lesion but studies showing association of RAS mutation with ovarian cancer is limited [101, 102]. RAS mutations in other form of human malignancies such as renal cancer [103], dermatological [104-107] and urinary bladder [108-110] have also reported.

6. Oncogenic RAS functioning machinery

RAS proteins are the vital component of the biological signaling pathways critical for growth, proliferation, differentiation, survival and death in almost all the organism ranging from yeast to humans. RAS proteins act as signal switch molecules for a plethora of mitogens including soluble growth factors by coupling the activated receptor to downstream effector pathways or proteins associated with the maintenance of normal cellular homeostasis including growth, proliferation, differentiation, survival and death [5, 6, 20]. Activation (RAS-GTP) and inactivation (RAS-GDP) of RAS proteins depend on the cooperative functioning of two factors i.e., GNEFs and GAPS. In general, mitogen or growth factor mediated activated receptor binds with intracellular signal relay molecules that results into recruitment and activation of GNEFs. GNEFs, in turn facilitate conversion of inactive (RAS-GDP) to active (RAS-GTP) form of RAS and this GTP-RAS stimulates functioning of a range of signaling pathways and effector molecules including RAF-MAPK/ERK kinase(MEK)- extracellular-signal-regulated kinas(ERK), phosphoinositide-3 kinase/protein kinase B (PI3K/AKT), phospholipase C (PLC)-protein kinase C (PKC), NORE1-RASSF1 (RAS association domain family member 1), RLDGSRAS-Like (RLDGS-RAL); and TIAMI-RAC-p21-activated kinase (PAK).
Under normal physiological conditions, RAS mediated activation of effectors is transient and tightly regulated by GAPs, which activates intrinsic GTP-ase activity of RAS and converts active RAS-GTP to inactive RAS-GDP state [20]. Deregulated expression and functioning of RAS is a well-known driving force for the genesis of most of the human pathological conditions including cancer due to mutation in RAS gene [111-113]. Indeed, it is well established that mutations in RAS genes along with the other genetic changes that activate regulators and effectors of RAS proteins are the prime cause of carcinogenesis and common in human cancers [20]. Aberrantly activated RAS become oncogenic by modulation of various signaling pathways and downstream effector molecules associated with the hallmarks of carcinogenesis. Here we have argued the role of RAF-MEK-ERK, PI3-AKT, PLC-PKC, NORE1-RASSF1 and RLDGS-RAL and TIAMI-RAC-PAK, signaling pathways in the RAS associated carcinogenesis.
RAS-GTP mediated activation of RAF-MEK-ERK signaling cascade is the most widely studied main effector pathway of RAS in response to stimuli associated with various cellular activities including proliferation, differentiation, and migration [113, 114].The RAS-RAF-MEK-ERK signaling pathway is one of the main mitogen-activated protein kinase (MAPK) cascades which translates message from various extracellular stimuli including growth factors, hormones, tumor promoting molecules, and differentiation factors into intracellular signals for regulating cell proliferation, differentiation, migration and survival by regulating the expression and functioning of various proteins [114-116].
RAS with intrinsic GTPase and kinases (RAF, MEK, and ERK) are the integral components in the RAS-RAF-MEK-ERK signaling machinery that work in response to stimuli. RAS mediated stimulation of RAF (ARF, BRAF and RAF1) serine/threonine kinases activate MEK-ERK kinase cascade resulting in the ERK mediated phosphorylation of a series of cytosolic and nuclear effector molecules including transcription factors (TFs) in tightly controlled fashion.
Oncogenic RAS induced dysregulation in RAS-RAF-MEK-ERK (MAPK/ERK pathway pathway) signaling pathway is one the prime mechanisms for RAS mediated carcinogenesis [11, 114] and the same has been detected in most of the human cancers. RAS mediated deregulation of RAF-MEK-ERK signaling cascade modulates expression and functioning of regulators and effectors of cellular processes such as proliferation, survival, differentiation and apoptosis to promote carcinogenesis [11, 114, 117].
A number of findings revealed that incessant RAS activation is the major driving force for uncontrolled cellular growth by modulating the expression and functioning of various nuclear TFs and cytosolic signaling molecules [118]. Recently, an investigation on how RAS-RAF-MEK-ERK pathway alters cellular metabolism to drive cell growth revealed that RAS signaling promotes RNA polymerase III (Pol III) function and t-RNA synthesis by inhibiting the Pol III repressor Maf1 [119].
In addition to the RAF-MEK-ERK pathway, RAS activates several other effector pathways, such as PI3K-AKT, PLC-PKC, NORE1-RASSF1, RLDGS-RAL and TIAMIRAC-PAK, as shown in fig.1. Another important downstream effector pathway critical for the mutagenic RAS triggered carcinogenesis is PI3K-AKT. RAS-GTP interaction with phosphatidylinositol 3-kinases (PI3Ks) leads to the activation of its kinase activity that phosphorylates phosphatidylinositol- 4,5-bisphosphate to produce phosphatidylinositol3,4,5-trisphosphate which acts as the second messenger and binds to various signaling moieties ranging from transcription factors to enzymes such as PDK1 (3phosphoinositide-dependent protein kinase-1) and AKT (protein kinase-B) [120, 121].PDK1 is important for the activation of various protein kinases including AKT/PKB, PKCs, p70S6K and RSK[120]. Activated AKT promotes cell survival by phosphorylation dependent activation and inactivation of survival and apoptotic proteins respectively [20].RAS-PI3-AKT signaling cascade is essential for the proper growth and cellular homeostasis [122, 123].
Oncogenic RAS induced deregulation of PI3K-AKT signaling cascade is essential for the carcinogenesis as it leads to the constitutive over expression of the regulator and effector molecules critical for cell growth and survival [122, 124, 125]. Another signaling cascade which gets affected due to oncogenic RAS is the RAL (RasLike) signaling pathway that plays important role in cellular biological processes essential for the survival and homeostasis [126-128]. The RAL-GTPase consists of the two family members (RALA and RALB) and their faction may be convergent or divergent in various biological conditions of cells including membrane ruffling, glycolysis, autophagy, secretion, cellular polarity, apoptosis and transcription. Aberrant expression or activation of RAL is critical in the process of carcinogenesis due to oncogenic RAS. RAS dependent activation of RAL signaling involves stimulation of various exchange factors like RALGDS, RALGDS-like gene (RGL) and RGL2 [126-128]. Once activated RAL can activate effectors like RALBP1 Sec5, Exo84,Filamin, PLD1 and ZONAB which play vital role in regulating expression and functioning of a range of transcription factors including, NF-kB, STAT3, and AP1[128]. There are various other direct effectors of RAS signaling cascade. RASSF5 (Ras association domain family member 5) also known as NORE1 is an important effector in RAS induced signaling associated with growth inhibition and apoptosis [129-131].

7. Oncogenic RAS and cancer hallmarks

RAS one the major oncogenes with neoplastic potential has been shown to play vital role in the development of characteristic features of cancer cells which are the major concern for present day cancer therapeutic measures. Uncontrolled cell proliferation, one of the main consequences of neoplastic transformation, is the main cause for expansion of cancer cells. RAS as an important signaling moiety decides the fate of mitogenic stimuli on cell proliferation and constitutive RAS activation is one of the main driving forces for limitless proliferative potential of cancer cells. Indeed, RAS itself has the potential to stimulate cell proliferation, activate transcriptional upregulation of growth factors, alteration in the functioning of growth factor receptors, integrin upregulation and inhibiting the anti-proliferative action and proteasomal degradation of cyclin dependent kinase inhibitors [12, 132-135]. There are various means by which RAS can activate limitless replication via controlling or altering the expression and or functioning of a battery of proteins associated with cell proliferation [12]. Apoptosis or programmed cell death is another important target for cancer treatment as the neoplastic cells did not respond to apoptotic signals and thus sustained with limitless proliferation. Oncogenic RAS play vital role in evasion of death signals or through regulation of signaling pathways such as PI3K and RAF [136].Limitless replication and differentiation of neoplasm requires abundant metabolic energy and oncogenic RAS is a central provider of power to the cells for proliferation through upregulation of hypoxia-inducible factor 1α (HIF1α), increased glucose transport via upregulation of glucose transporter GLUT1, activating glycolytic pathways and enzymes and also through regulation of autophagy [137-144]. Oncogenic RAS has emerged as an important driving force in angiogenesis or formation of new blood vessels which is vital for survival and growth of neoplasm as the main provider of oxygen and nutrients [145-147]. Another important issue of cancer cell is their insensitivity towards host immune response which ideally should recognize tumor as foreign entity and sabotage cancer cells. Oncogenic RAS is integral in enabling immune surveillance insensitive towards the cancer cells as a threat or danger via modulating the expression of antigen-presenting major histocompatibility complexes (MHC) and also through attenuation of host immune response by making T cells friendly attitude [145-148]. Progression, metastasis or spreading of cancer cells towards other body parts is the most serious feature of carcinogenesis. Mutated RAS is central in the processes associated with cancer cell invasion and migration [149-151].Very recently, it has been revealed that Myc in cooperation with RAS triggers carcinogenesis (cancer migration and metastasis) by reprogramming tumor micro-environment via immunosuppression [151].

8. Oncogenic RAS and transcription factors (TFs)

Transcription factors (TFs), are the specific DNA sequence binding proteins which regulate gene transcription by acting on DNA-regulatory sequences (promoters and silencers), can increase or decrease the expression of genes and thus play vital role in development and functioning of the biological system. Significant proportion of human genome encodes over 2000 different TFs which are the main regulators of gene expression in all 17 major signal transduction and two stress response pathways [152, 153]. Dysregulation in the expression and functioning of (TFs) is generally considered as the driving force for development of various pathological conditions including cancer [153, 154].
Oncogenic mutation induces constitutive activation of signaling pathways which in turn modulate functioning of transcription factors that regulate various genes associated with the hallmarks of carcinogenesis [154] (fig.2). Mutated RAS mediated cellular transformation requires dysregulation of various signaling effectors including TFs [12] . Nuclear factor kappa-B (NF-kB), a key transcription factor vital for proper functioning and homeostasis of cell, is a central player in carcinogenesis including oncogenic RAS induced cancer development [155-158]. Normally NF-kB mediated transcriptional expression of genes is associated with host immune response and inflammation but mutation in its upstream signaling components such as RAS results into dysregulated expression and functioning which in turn accelerate expression of genes associated with carcinogenesis or neoplastic transformation [155-158].
Various reports have documented the vital role of NF-kB in RAS induced cancer development. For instance, in WB rodents epithelial cell, RAS induced malignant progression require NF-kB mediated upregulation of COX-2 and MMP9 [159]. NF-kB regulates the expression of interleukins which are critical for RAS induced cellular transformation and tumorigenesis [160, 161]. Indeed there is a connection between oncogenic RAS and NF-kB in cancer development as evident from the findings that
RAS induced lung adenocarcinoma require NF-kB activation and inhibition of NF-kB causes cell death and regression in tumor growth [155, 162]. Inhibition of NF-kB activation in cancer cells with oncogenic RAS by lysyl oxidase provides another evidence for the critical role of NF-kB in RAS associated oncogenesis [163] . Cancer associated resistance is a major concern of present day cancer therapy, inhibition of NF-kB radio sensitizes Ki-RAS-transformed cells to ionizing radiation which reflects therapeutic role of NF-kB inhibition in RAS associated neoplasms [164].In another study, it was found that oncogenic RAS induced endometrial carcinogenesis require activation of NF-kB mediated transcription of signaling molecules suggesting the role of NF-kB in the pathogenesis of RAS induced endometrial cancer and its targeted therapy [165].
Signal transducer and activator of transcription (STAT) proteins, a family of seven transcription factors, are the well-known molecules required for proper functioning and maintenance of cellular and biological integrity as they act both as signal transducers as well as transcription factors for the expression and functioning of battery of genes. An array of scientific investigations show that dysregulation of STATs particularly STAT3 and to some extent STAT5 play major role in carcinogenesis via contributing in initiation and progression by inducing proliferation, survival, angiogenesis, inhibiting anti-tumor immune responses, and resistance of tumor cells [166-169].
Important role of STATs in the oncogenic RAS associated carcinogenesis is evident from various preclinical and clinical research investigations. In relation to this, STAT3 has been shown to play major role in growth and survival of oncogenic RAS transformed intestinal epithelial cells, fibroblast and breast cancer cells by augmenting tyrosine kinase activity and upregulating apoptosis inhibitory signals [170, 171]. In case of familial medullary thyroid carcinoma (FMTC) activation of a Ras/ERK1/2/STAT3 pathway is required for cell transformation and tumor cell proliferation [172]. Interestingly, in a double conditional transgenic tumor model of lung adenocarcinoma and lymphoma study it was found that there is a strong association between oncogenic RAS and STAT signaling proteins critical in the development of both lung tumors and lymphomas [173]. In another study, in mouse fibroblast and xenograft mouse, over expression of phosphorylated STAT3 by RAS showed higher colony formation and tumor growth [174]. Role of STAT3 in RAS induced cellular transformation and carcinogenesis is not only restricted to transcriptional dysregulation of genes but also regulates a metabolic function in mitochondria in sustaining the altered glycolytic and oxidative phosphorylation activities [175]. In a chemically induced mouse model of skin carcinogenesis, RAS induced activation of STAT3 is necessary for the initiation phase of cancer development in the keratinocyte stem cells [176]. In case of oncogenic RAS induced pancreatic ductal adenocarcinoma (PDA), role of STAT3 is critical in initial precancerous lesions, promotion and neoplastic changes (acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) [177, 178] and recently the role of STAT5 in RAS associated PDA is also reported [179]. Furthermore, there is a strong correlation between oncogenic RAS and STAT3 functioning in the development of hematological malignancies including Juvenile myelomonocytic leukemia, acute myeloid leukemia (AML), and other myeloproliferative neoplasms (MPNs) as STAT3 play critical role in the RAS driven proliferation of myeloid cells [180]. In another study, transformation of metaplastic epithelial cells of Barrett’s esophagus in combination with p53-knockdown and RAS activation has shown that mitochondrial STAT3 can contribute to oncogenesis in Barrett’s cells that express oncogenic Ras [181]. STAT3 acts as a mediator that synergizes TGF-β and Ras signals through induction of Snail, a key regulator of epithelial-mesenchymal transition, crucial morphological event that occurs during the progression of epithelial tumors [182].
Forkhead box protein M1 (FOXM1, other aliases, HFH-11B, Trident, Win, or MPP2), another important component in RAS mediated oncogenesis, is a TF of highly conserved forkhead family of transcription factors which is known for its regulatory action on various biological activities such as proliferation, cell cycle, differentiation, genomic repair, angiogenesis and programmed cell death[183-185]. Various documented findings have revealed that dysregulation in FOXM1 play critical role in oncogenic RAS mediated carcinogenesis of various organs. An experimental study on transgenic mouse provides genetic evidence that FoxM1 is essential for RAS driven progression of hepatic cancer [186]. Another study on oncogenic RAS associated gene signature analysis in 55 colorectal cancer (CRC) bulk tumors revealed enriched expression of FOXM1 showing its important role as reduction in FOXM1 expression was found to be sufficient to inhibit growth and reduce gene enrichment in RAS associated CRC [187]. Oxidative stress or over production of ROS (reactive oxygen species) play major role in oncogenic RAS induced carcinogenesis via regulating the expression of FOXM1 as RAS causes release of ROS which in turn upregulate FOXM1 that functions in a negative feedback manner and protect cells via upregulating antioxidant enzymes and thus promotes survival [188]. These observations suggest FOXM1 is critical for growth and survival of tumor cells harboring oncogenic RAS [188]. Clinically, overexpression of FOXM1 in oncogenic RAS induced lung carcinomas is often associated with poor prognosis. Being an important downstream target of RAS, role of FOXM1 in initiation of oncogenic RAS induced tumorigenesis was delineated recently [189]. In fact, deletion of FOXM1 in lung epithelial cells inhibits oncogenic RAS induced lung tumorigenesis via preventing RAS mediated activation of genes critical for the activation of JNK and NF-Kb signaling pathways [189].
Another transcription factor which play important role in oncogenic RAS associated tumorigenesis is the activator protein 1 (AP1) as it regulate the expression and functioning of genes associated with cell growth, proliferation, transformation, survival and apoptosis. AP1 TF is a dimeric complex that consists of JUN, FOS, ATF and MAF protein family members and thus can form various combinations of heterodimers and homodimers which decide the genes that are regulated by AP-1[190]. Various findings demonstrated that upregulation of AP1 associated proteins is critical for RAS induced tumorigenesis as cellular transformation of RAS requires upregulation of AP1 activity to promote cell proliferation [191]. Very recently, it was found that one of the long noncoding RNAs (lncRNA) named lncRNA Orilnc1 is critical in RAS induced cellular transformation and AP1 plays vital role in the expression of lncRNA Orilnc1 [192].Interestingly it was also observed that mutagenic RAS induced lung tumor formation requires JNK mediated activation of AP TFs further supported the role of AP1 TFs in mutagenic RAS associated cancer development [193].
Other TFs that play vital role in RAS associated carcinogenesis by dysregulation in the expression and transcriptional functioning of array signaling proteins includes hypoxia inducible factor-1a (HIF-1A) and E26 transformation-specific (Ets). Various findings revealed that oncogenic RAS activates HIF-1a, which play key role in metabolic reprograming, proliferation, angiogenesis, and survival of cancer cells [138, 194-197]. Recently Etv5, a member of Ets family of transcription factors, has been shown to play vital role in RAS induced lung tumorigenesis while the role of other Ets family members in oncogenic RAS signaling is well known as RAS induced dysregulation in Ets functioning alters normal cell functions and promotes unlimited increased proliferation, sustained angiogenesis, invasion and metastasis [198, 199].

9. RAS and Inflammation

Inflammation, one of the most discussed phenomenons in carcinogenesis, is generally a defense mechanism but becomes life threatening when deregulated. Due to its well established role in oncogenesis, malignant transformation, tumor growth, tumor invasion, metastasis, resistance, inflammation has now been considered as an important cancer hallmark [200, 201].RAS being an oncogenic addiction gene, critically associated with the maintenance of tumor microenvironment inflammation by triggering persistent activation of various inflammatory mediators including TFs, inflammatory cells, cytokines, chemokines and growth factors [202-204]. In this context, investigations on how oncogenic RAS activate the upstream regulators or inducers of inflammatory moieties have been well explored [203, 204]. In the next section of this review, we have argued in detail about the role of NF-kB, the master regulator of inflammation, and STAT3 in oncogenic RAS induced cancer development. RAS signaling cascade promote the expression of cytokines, chemokines and adhesion molecules in building inflammatory microenvironments that contribute to tumor growth and invasion via modifying ingredients (fibroblasts, immune and inflammatory cells, adipocytes, and endothelial cells) of tumor stroma [161, 205-207]. While it has been well explained that oncogenic RAS signaling can modulate the expression of various inflammatory mediators including NF-KB which is vital for the tumorigenesis and cytokine release but the underlying mechanism requires further attention. Recently, it has been revealed that oncogenic RAS signaling activates ETS transcription factor PEA3 mediated transcriptional upregulation of squamous cell carcinoma antigens 1 and 2 (SCCA1/2) (members of the Serpin family of serine/cysteine protease inhibitors) which in turn leads to inhibition of protein turnover, unfolded protein response, activation of NF-κB suggesting that SCCA as a direct link between oncogenic RAS mutation, NF-κB activation, and cytokine production [208]. Giannou et al [209] recently discovered that inoculation of tumor cells of various tissues of origin harboring N-RAS mutations are able to spontaneously metastasize to the lungs of mice via upregulating interleukin-8related chemokine (CXCR1/2) signaling. In light of the important role of inflammation in RAS associated malignancies further investigations are needed for precise therapeutic targets.

10. Oncogenic RAS signaling and F-box protein SKP2 expression

Oncogenic RAS mediated cancer development involves modulation of a plethora of regulatory proteins known as the central regulator of normal cellular homeostasis. Considering this notion here we have argued the oncogenic relation between RAS and S-phase associated protein kinase 2 (SKP2). Basically it is a member of F-box family of substrate-recognition subunits of SCF ubiquitin–protein ligase complexes that regulates the periodic concentration of various regulatory proteins (e.g., p27, p21, cyclin E1 etc.) to maintain the highly ordered and controlled progression of cell cycle by inducing polyubiquitination and degradation [210]. Over expression of Skp2 is ubiquitous in human cancers and is also associated with poor prognosis [211]. Considering its vital role in cancer development, SKP2 is now known as onco-protein.
Indeed, available research findings reflect that there is a strong oncogenic relation between RAS and SKP2 protein mediated polyubiquitination of regulatory proteins. Recently, it has been shown that oncogenic RAS upregulate SKP2 mediated polyubiquitination and activation of LKB1, a tumor suppressor kinase well known for its vital role in various cellular activities like, proliferation, energy regulation, polarity and death, which is essential for the cancer cell survival under energy stress via activation of the SKP2-SCF complex [212].
Further, another interesting report of cooperative functioning of oncogenic RAS and SKP2 have shown that over expression of SKP2 in mouse liver alone did not induce tumor formation while its co-expression with oncogenic form of human RAS oncogenes (N-RasV12) results into hepatocarcinogenesis [213]. There are other reports which revealed that proto-oncoprotein SKP2 needs RAS cooperation to induce cellular transformation and carcinogenesis [210, 214]. Growth inhibitory action of TGF-beta on the proliferation of hematopoietic cells with RAS mutation rendered ineffective due to SKP2 mediated polyubiquitination and degradation of cyclin dependent kinase inhibitor p27 in a Ras/MEK/Erk/SKP2-dependent pathway [215]. Over all, it has become evident that there is a strong co-operative oncogenic functioning relation between RAS and SKP2 and targeting the RAS-RAF-MEK-ERK-SKP2 axis may serve as an important target for cancer therapy.

11. RAS signaling in cancer stem cells

The presence of a rare subpopulation called cancer stem cell (CSC) within the bulk of heterogeneous cancer cell population and their role in cancer aggressiveness, metastatic potential, resistance to therapy and rate of relapse has been confirmed in the last decade [216]. The existence of CSCs characterized by self-renewal capacity, unlimited repopulating potential and a quiescent nature, was pointed out by Lapidot el al [217] in acute myeloid leukemia [218]. Later the concept of CSCs was confirmed in various other hematological malignancies, followed by a wide range of epithelial and solid organ cancers including liver, brain, breast, lung, melanoma, colon, etc., which are shown to have a unique surface marker signature depending on the cancer type. Currently CSCs are considered as a novel druggable target for the treatment of various cancers.
In colorectal cancer, gain-of-function mutations in K-RAS were found to activate CSC population, which contributes to colorectal tumorigenesis and metastasis. Loss-offunction mutations in the tumor suppressor gene, adenomatous polyposis coli (APC), are needed for this RAS induced CSC activation. Initial activation of β-catenin by APC loss induces expression of colorectal cancer stem cell markers such as CD44, CD133, and CD166 which is further enhanced by K-RAS mutation [219]. In colorectal cancer patients mutations in APC and K-RAS are common abnormalities in the initial and intermediate adenoma stages.
Oncogenic K-RAS can promote proliferative and self-renewal properties of quiescent intestinal stem cells which are dependent upon activation of mitogen-activated protein kinase (MEK)[220]. Self-renewing tumor-initiating cells has been known to contribute to tumor progression, recurrence and resistance in breast cancer. Liu et al [221] reported that p21CIP1 can attenuate RAS dependent epithelial mesenchymal transition and CSC like gene expression in breast cancer. Loss of p21CIP1 leads to enhancement of mammary-targeted Ha-RAS induced tumorigenesis. Let-7a, belonging to the first identified tumor suppressing miRNA family Let-7, was found to have reduced expression in several solid tumors, including breast cancer patient samples. Let-7a inhibited mammosphere-forming efficiency and the mammosphere-size through NF-κB and MAPK/ERK pathway by inhibition of oncogenic RAS [222].
In a mouse model of breast cancer, RAS activation was found to play a vital role in the maintenance and expansion of stem cell antigen-1 (Sca-1) positive cells through modulation of expression of stem cell transcription factors such as Sox2, Oct-4 and Nanog [223].
RAS superfamily member Rac, belonging to Rho GTPase subfamily, is involved in the maintenance of glioma stem-like cell population and tumorigenicity, through hyperactivation of Rac1-Pak signaling pathway. Knockdown/downregulation of Rac1 leads to inhibition of glioma stem-like markers, proteins involved in self-renewal and proliferation (Sox2, Notch1/2, and β-catenin) and neurosphere formation. Rac1 mediated signaling was found to be vital for glioma stem-like cells for resistance to anticancer treatments [224].
Tumors of endodermal origin (lung, pancreas, and colon) have high frequency of activating mutations in K-RAS. Activation of K-RAS was found to initiate tumors of endodermal origin through the expansion of stem cell. In an in vitro model of retinoic acid -induced stem cell differentiation to endoderm, activation of HrasV12 was found to promote differentiation and growth arrest, while activation of KrasV12 inhibited retinoic acid induced differentiation, and promote proliferation and maintenance of stem cell characteristics in endodermal progenitors. Activation of NrasV12 had no effect on differentiation of endodermal progenitors [225].
Approximately 10% of non-small cell lung cancer cases are found to have activating mutations in K-RAS, while more than 30% adenocarcinomas have K-RAS mutations. In a mouse model of oncogenic K-RAS-induced lung adenocarcinoma, phosphatidylinositol 3-kinase was found to mediate the expansion of bronchio-alveolar stem cells, a putative cancer stem cell population. Inactivation of PTEN, a negative regulator of PI3K, was found to enhance K-RAS induced stem cell accumulation [226].
Pancreatic adenocarcinomas usually have a very poor prognosis, partly due to resistance to gemcitabine, which is the first line treatment. Gemcitabine treatment was found to induce metabolic reprogramming from oxidative phosphorylation towards glycolysis and enhancement of cancer stem-like status. Protein and mRNA levels of Nanog and Sox2 were increased with gemcitabine treatment along with increase in expression of pancreatic stem-like cells markers (ALDH, OCT4, KLF4, CXCR4, and CD24). Drug resistance related genes (ABCG2 and MDR1), differentiation markers (ID1, MUC-1 and MUC-4) and epithelial-mesenchymal transition -associated genes (Ncadherin, Vimentin, Snail1, and Zeb1) were also upregulated with gemcitabine treatment of various pancreatic cancer cell lines. Gemcitabine exposure activated KRas, which is involved in the induction of metabolic reprogramming and cancer cell stemness through regulation of AMPK. Okada et al reported that knockdown of K-RAS in pancreatic stem-like cells with activating K-RAS mutation led to the loss of selfrenewal and tumor-initiating capacity, via the inhibition of JNK [227].
Overexpression of the constitutively active RAS-V12 induces senescence in transientamplifying primary human keratinocytes expressing high levels of p16, while RAS overexpression in highly proliferative stem-cell-enriched cultures with low levels of p16 sustains indefinite culture growth. Evading senescence correlates with retinoblastoma pathway inhibition and restoration of telomerase reverse transcriptase activity, and the immortalization is maintained by the ERK1/2 and Akt pathways [228]. Recent studies showed that HCC patients have ubiquitous activation of the RAS signaling pathway through epigenetic silencing of the RAS-regulators such as RASSF1A, NORE1A/B and Ras GTPase-activating proteins. Overexpression of FoxM1, a fork-head box family transcription factor, in HCC correlated with poor prognosis. In K-RAS overexpressing tumors, FoxM1 activates the expression of stem cell marker CD44 and drives the stem cell features and tumor progression. The CSC population needed FoxM1 for ROSregulation and survival [186].

12. Oncogenic RAS signaling and immune check point inhibitor deregulation

Evasion of host immune checkpoint, a principal hallmark of cancer cells is one of the critical issues associated with the carcinogenesis and therapeutic interventions. Cancers harboring over expressed RAS or its regulators are not being able to be recognized as foreign entity by the host immune system. Various findings revealed that oncogenic RAS mediated activation of signaling cascade play major role in the impairment of the host antitumor immune response via modulating the expression as well as functional regulation of immune cells, immunosuppressive cytokines, chemokines and their regulators [229, 230]. Here we have discussed the role of oncogenic RAS signaling in subverting anti-tumor immunity by modulating the expression and functioning of programmed death-ligand 1 (PD-L1) and its regulatory molecules. PD-L1 also known as cluster of differentiation 274 (CD274), B7 homolog 1 (B7-H1), is a transmembrane protein and well known for its immunosuppressive potential [231]. Human cancer cells often detected with high PD-L1 expression that impair the killing potential of immune cells such as B, T and NK cells and thus considered as the main contributor in the immune escape of cancer cells [232, 233]. An array of preclinical and clinical investigations have explained that oncogenic RAS signaling cascade play a major role in the immune escape of cancer cells by inducing the upregulation in PD-L1 expression[233]. In this context, a very recent study revealed that over expression of PD-L1 in lung adenocarcinoma is due to the activation of mutagenic RAS signaling cascade and also found that RAS-mediated up-regulation of PD-L1 induced the apoptosis of CD3+ T cells and mediated immune escape in lung adenocarcinoma cells [234]. One of the possible mechanisms involved in the oncogenic RAS induced tumor-immune resistance is most likely through enhancing PD-L1 mRNA stability via modulation/inhibition of the AU-rich element-binding protein tristetraprolin (TTP), a negative regulator of PD-L1 expression [19]. Thus development of strategies to block RAS mediated upregulation of PD-L1 may provide an important option for cancer therapy.

13. Conclusions and future directions

RAS is an oncogenic driver that makes it an important molecular target for the treatment of various human malignancies. Decades of intensive hard work on RAS in normal physiology and in various pathological conditions including cancer by using various possible experimental approaches to understand how and when RAS become dangerous and act as the driving force for the genesis of pathological conditions has explored various attitude of RAS that can accelerate disease development. One of the major driving forces behind RAS induced disease development like cancer is the mutation in the genetic content of RAS and its regulator genes. Mutated RAS genes encodes hyperactive RAS proteins which lead to the dysregulation in the expression and functioning of all the major signaling cascade mechanisms intricately involved in the maintenance of cellular homeostasis contributes to its oncogenic potential. With the use of modern genetic tools for detecting mutation in various human diseases including cancer has revealed that a significant proportion of most of the human cancers are harboring mutation in RAS gene. Here in this review we have argued how the oncogenic RAS act as a driving force in carcinogenesis and in the inadequate therapeutic outcomes. Mutations in RAS genes latches it in the GTP-bound form and not allowing GDP and GTP cycle to proceed as it occur with normal RAS. Hyperactive oncogenic RAS causes incessant dysregulation in major signaling cascade such as RAF-MEK-ERK, MAP kinase, PI3-kinase/AKT that can help in sustained survival and proliferation of cancer cells proliferation and other aggressive changes to the cells that support the malignant phenotype. Oncogenic RAS induced aberrant dysregulation of signaling cascade modulates functioning of array of downstream effectors which ultimately lead to unregulated transcriptional expression and functioning of genes associated with the carcinogenesis. Intense research work of decades has revealed that aberrant functioning of TFs is the main effectors in oncogenic RAS mediated cancer development and its implication in attaining cancer hallmarks. Targeting oncogenic RAS in various forms of experimental models have greatly contributed to the understanding of RAS signaling pathways in carcinogenesis and a number of preclinical and clinical studies suggested that RAS can be a therapeutic target for cancer eradication. Recent evidences related to the involvement of RAS in the emerging hallmark of cancer suggest that development of oncogenic RAS specific inhibitors could be promising approach for therapeutic intervention of RAS associated malignancies. In spite of the enormous development of various effective possible ways in search of inhibiting RAS mediated signaling dysregulation we are still lacking behind in the effective management of RAS associated neoplasms. Furthermore, inadequate clinical outcomes in RAS associated malignancies indicate that there are various lacunas in several points in understanding RAS biology, its dynamics and associated signaling which needs further attention which will greatly help in developing novel strategies for therapeutics in targeting RAS and associated signaling effectors in human malignancies.

References:

[1]Rojas AM, Fuentes G, Rausell A, Valencia A. The Ras protein superfamily: evolutionary tree and role of conserved amino acids. J Cell Biol. 2012;196:189-201.
[2]Wennerberg K, Rossman KL, Der CJ. The Ras superfamily at a glance. J Cell Sci. 2005;118:843-6.
[3]Vetter IR, Wittinghofer A. The guanine nucleotide-binding switch in three dimensions. Science. 2001;294:1299-304.
[4]Ward AF, Braun BS, Shannon KM. Targeting oncogenic Ras signaling in hematologic malignancies. Blood. 2012;120:3397-406.
[5]Vigil D, Cherfils J, Rossman KL, Der CJ. Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? Nat Rev Cancer. 2010;10:842-57.
[6]Zhou B, Der CJ, Cox AD. The role of wild type RAS isoforms in cancer. Semin Cell Dev Biol. 2016;58:60-9.
[7]Mann KM, Ying H, Juan J, Jenkins NA, Copeland NG. KRAS-related proteins in pancreatic cancer. Pharmacol Ther. 2016;168:29-42.
[8]Croce CM. Oncogenes and cancer. N Engl J Med. 2008;358:502-11.
[9]LeBleu VS, Taduri G, O’Connell J, Teng Y, Cooke VG, Woda C, Sugimoto H, Kalluri R. Origin and function of myofibroblasts in kidney fibrosis. Nat Med. 2013;19:1047-53.
[10]Logsdon CD, Lu W. The Significance of Ras Activity in Pancreatic Cancer Initiation. Int J Biol Sci. 2016;12:338-46.
[11]Samatar AA, Poulikakos PI. Targeting RAS-ERK signalling in cancer: promises and challenges. Nat Rev Drug Discov. 2014;13:928-42.
[12]Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer. 2011;11:761-74.
[13]Baines AT, Xu D, Der CJ. Inhibition of Ras for cancer treatment: the search continues. Future Med Chem. 2011;3:1787-808.
[14]Rodenhuis S. ras and human tumors. Semin Cancer Biol. 1992;3:241-7.
[15]Mengoli MC, Barbieri F, Bertolini F, Tiseo M, Rossi G. K-RAS mutations indicating primary resistance to crizotinib in ALK-rearranged adenocarcinomas of the lung: Report of two cases and review of the literature. Lung Cancer. 2016;93:55-8.
[16]Beaupre DM, Kurzrock R. RAS and leukemia: from basic mechanisms to genedirected therapy. J Clin Oncol. 1999;17:1071-9.
[17]Bahrami A, Hesari A, Khazaei M, Hassanian SM, Ferns GA, Avan A. The therapeutic potential of targeting the BRAF mutation in patients with colorectal cancer. J Cell Physiol. 2018;233:2162-9.
[18]Martinelli E, Morgillo F, Troiani T, Ciardiello F. Cancer resistance to therapies against the EGFR-RAS-RAF pathway: The role of MEK. Cancer Treat Rev. 2017;53:61-9.
[19]Coelho MA, de Carne Trecesson S, Rana S, Zecchin D, Moore C, Molina-Arcas M, East P, Spencer-Dene B, Nye E, Barnouin K, Snijders AP, et al. Oncogenic RAS Signaling Promotes Tumor Immunoresistance by Stabilizing PD-L1 mRNA. Immunity. 2017;47:1083-99 e6.
[20]Schubbert S, Shannon K, Bollag G. Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer. 2007;7:295-308.
[21]Chin L, Tam A, Pomerantz J, Wong M, Holash J, Bardeesy N, Shen Q, O’Hagan R, Pantginis J, Zhou H, Horner JW, 2nd, et al. Essential role for oncogenic Ras in tumour maintenance. Nature. 1999;400:468-72.
[22]Hobbs GA, Der CJ, Rossman KL. RAS isoforms and mutations in cancer Darovasertib at a glance. J Cell Sci. 2016;129:1287-92.
[23]Knickelbein K, Zhang L. Mutant KRAS as a critical determinant of the therapeutic response of colorectal cancer. Genes Dis. 2015;2:4-12.
[24]Karnoub AE, Weinberg RA. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol. 2008;9:517-31.
[25]Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779-827.
[26]Cohen JB, Broz SD, Levinson AD. Expression of the H-ras proto-oncogene is controlled by alternative splicing. Cell. 1989;58:461-72.
[27]Seeburg PH, Colby WW, Capon DJ, Goeddel DV, Levinson AD. Biological properties of human c-Ha-ras1 genes mutated at codon 12. Nature. 1984;312:71-5.
[28]Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012;72:2457-67.
[29]Bos JL. ras oncogenes in human cancer: a review. Cancer Res. 1989;49:4682-9. [30] Parsons BL, Culp SJ, Manjanatha MG, Heflich RH. Occurrence of H-ras codon 61 CAA to AAA mutation during mouse liver tumor progression. Carcinogenesis. 2002;23:943-8.
[31]Smith G, Bounds R, Wolf H, Steele RJ, Carey FA, Wolf CR. Activating K-Ras mutations outwith ‘hotspot’ codons in sporadic colorectal tumours – implications for personalised cancer medicine. Br J Cancer. 2010;102:693-703.
[32]Johnson L, Mercer K, Greenbaum D, Bronson RT, Crowley D, Tuveson DA, Jacks T. Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature. 2001;410:1111-6.
[33]Stolze B, Reinhart S, Bulllinger L, Frohling S, Scholl C. Comparative analysis of KRAS codon 12, 13, 18, 61, and 117 mutations using human MCF10A isogenic cell lines. Sci Rep. 2015;5:8535.
[34]Phipps AI, Buchanan DD, Makar KW, Win AK, Baron JA, Lindor NM, Potter JD, Newcomb PA. KRAS-mutation status in relation to colorectal cancer survival: the joint impact of correlated tumour markers. Br J Cancer. 2013;108:1757-64.
[35]Imamura Y, Morikawa T, Liao X, Lochhead P, Kuchiba A, Yamauchi M, Qian ZR, Nishihara R, Meyerhardt JA, Haigis KM, Fuchs CS, et al. Specific mutations in KRAS codons 12 and 13, and patient prognosis in 1075 BRAF wild-type colorectal cancers. Clin Cancer Res. 2012;18:4753-63.
[36]Edkins S, O’Meara S, Parker A, Stevens C, Reis M, Jones S, Greenman C, Davies H, Dalgliesh G, Forbes S, Hunter C, et al. Recurrent KRAS codon 146 mutations in human colorectal cancer. Cancer Biol Ther. 2006;5:928-32.
[37]Omholt K, Karsberg S, Platz A, Kanter L, Ringborg U, Hansson J. Screening of Nras codon 61 mutations in paired primary and metastatic cutaneous melanomas: mutations occur early and persist throughout tumor progression. Clin Cancer Res. 2002;8:3468-74.
[38]Irahara N, Baba Y, Nosho K, Shima K, Yan L, Dias-Santagata D, Iafrate AJ, Fuchs CS, Haigis KM, Ogino S. NRAS mutations are rare in colorectal cancer. Diagn Mol Pathol. 2010;19:157-63.
[39]Quilliam LA, Castro AF, Rogers-Graham KS, Martin CB, Der CJ, Bi C. M-Ras/RRas3, a transforming ras protein regulated by Sos1, GRF1, and p120 Ras GTPaseactivating protein, interacts with the putative Ras effector AF6. J Biol Chem. 1999;274:23850-7.
[40]Matsumoto K, Asano T, Endo T. Novel small GTPase M-Ras participates in reorganization of actin cytoskeleton. Oncogene. 1997;15:2409-17.
[41]Huff SY, Quilliam LA, Cox AD, Der CJ. R-Ras is regulated by activators and effectors distinct from those that control Ras function. Oncogene. 1997;14:133-43.
[42]Fernandez-Medarde A, Santos E. Ras in cancer and developmental diseases. Genes Cancer. 2011;2:344-58.
[43]Caldas C, Hahn SA, Hruban RH, Redston MS, Yeo CJ, Kern SE. Detection of K-ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia. Cancer Res. 1994;54:3568-73.
[44]Lu L, Zeng J. Evaluation of K-ras and p53 expression in pancreatic adenocarcinoma using the cancer genome atlas. PLoS One. 2017;12:e0181532. [45] Lee JH, Kim Y, Choi JW, Kim YS. KRAS, GNAS, and RNF43 mutations in intraductal papillary mucinous neoplasm of the pancreas: a meta-analysis. Springerplus. 2016;5:1172.
[46]Hashimoto D, Arima K, Yokoyama N, Chikamoto A, Taki K, Inoue R, Kaida T, Higashi T, Nitta H, Ohmuraya M, Hirota M, et al. Heterogeneity of KRAS Mutations in Pancreatic Ductal Adenocarcinoma. Pancreas. 2016;45:1111-4.
[47]Miglio U, Oldani A, Mezzapelle R, Veggiani C, Paganotti A, Garavoglia M, Boldorini R. KRAS mutational analysis in ductal adenocarcinoma of the pancreas and its clinical significance. Pathol Res Pract. 2014;210:307-11.
[48]Sinn BV, Striefler JK, Rudl MA, Lehmann A, Bahra M, Denkert C, Sinn M, Stieler J, Klauschen F, Budczies J, Weichert W, et al. KRAS mutations in codon 12 or 13 are associated with worse prognosis in pancreatic ductal adenocarcinoma. Pancreas. 2014;43:578-83.
[49]Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, Jia M, Shepherd R, Leung K, Menzies A, Teague JW, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 2011;39:D945-50. [50] Immervoll H, Hoem D, Kugarajh K, Steine SJ, Molven A. Molecular analysis of the EGFR-RAS-RAF pathway in pancreatic ductal adenocarcinomas: lack of mutations in the BRAF and EGFR genes. Virchows Arch. 2006;448:788-96.
[51]Kawesha A, Ghaneh P, Andren-Sandberg A, Ograed D, Skar R, Dawiskiba S, Evans JD, Campbell F, Lemoine N, Neoptolemos JP. K-ras oncogene subtype mutations are associated with survival but not expression of p53, p16(INK4A), p21(WAF-1), cyclin D1, erbB-2 and erbB-3 in resected pancreatic ductal adenocarcinoma. Int J Cancer. 2000;89:469-74.
[52]Whyte DB, Kirschmeier P, Hockenberry TN, Nunez-Oliva I, James L, Catino JJ, Bishop WR, Pai JK. K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem. 1997;272:14459-64.
[53]Weden S, Klemp M, Gladhaug IP, Moller M, Eriksen JA, Gaudernack G, Buanes T. Long-term follow-up of patients with resected pancreatic cancer following vaccination against mutant K-ras. Int J Cancer. 2011;128:1120-8.
[54]Boutin AT, Liao WT, Wang M, Hwang SS, Karpinets TV, Cheung H, Chu GC, Jiang S, Hu J, Chang K, Vilar E, et al. Oncogenic Kras drives invasion and maintains metastases in colorectal cancer. Genes Dev. 2017;31:370-82.
[55]Poulin EJ, Haigis KM. No back seat for a progression event-K-RAS as a therapeutic target in CRC. Genes Dev. 2017;31:333-5.
[56]Abubaker J, Bavi P, Al-Haqawi W, Sultana M, Al-Harbi S, Al-Sanea N, Abduljabbar A, Ashari LH, Alhomoud S, Al-Dayel F, Uddin S, et al. Prognostic significance of alterations in KRAS isoforms KRAS-4A/4B and KRAS mutations in colorectal carcinoma. J Pathol. 2009;219:435-45.
[57]Lievre A, Merlin JL, Sabourin JC, Artru P, Tong S, Libert L, Audhuy F, Gicquel C, Moureau-Zabotto L, Ossendza RA, Laurent-Puig P, et al. RAS mutation testing in patients with metastatic colorectal cancer in French clinical practice: A status report in 2014. Dig Liver Dis. 2018.
[58]Petaccia de Macedo M, Melo FM, Ribeiro HSC, Marques MC, Kagohara LT, Begnami MD, Neto JC, Ribeiro JS, Soares FA, Carraro DM, Cunha IW. KRAS mutation status is highly homogeneous between areas of the primary tumor and the corresponding metastasis of colorectal adenocarcinomas: one less problem in patient care. Am J Cancer Res. 2017;7:1978-89.
[59]Zekri J, Al-Shehri A, Mahrous M, Al-Rehaily S, Darwish T, Bassi S, El Taani H, Al Zahrani A, Elsamany S, Al-Maghrabi J, Sadiq BB. Mutations in codons 12 and 13 of Kras exon 2 in colorectal tumors of Saudi Arabian patients: frequency, clincopathological associations, and clinical outcomes. Genet Mol Res. 2017;16.
[60]Kafatos G, Niepel D, Lowe K, Jenkins-Anderson S, Westhead H, Garawin T, Traugottova Z, Bilalis A, Molnar E, Timar J, Toth E, et al. RAS mutation prevalence among patients with metastatic colorectal cancer: a meta-analysis of real-world data. Biomark Med. 2017.