Seminars in Cancer Biology xxx (xxxx) xxx–xxx

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Seminars in Cancer Biology

journal homepage: www.elsevier.com/locate/semcancer

Review

RAS mutations in human cancers: Roles in precision medicine

Avaniyapuram Kannan Murugana, , Michele Griecob, Nobuo Tsuchidaa,
⁎ ⁎⁎

a
Department of Molecular Cellular Oncology and Microbiology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 Japan
b
DiSTABiF, Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università di Napoli, via Vivaldi 43, Caserta 81100 Italy

ARTICLE INFO ABSTRACT

Keywords: Ras proteins play a crucial role as a central component of the cellular networks controlling a variety of signaling
Mutation pathways that regulate growth, proliferation, survival, differentiation, adhesion, cytoskeletal rearrangements
Precision medicine and motility of a cell. Almost, 4 decades passed since Ras research was started and ras genes were originally
Oncogene discovered as retroviral oncogenes. Later on, mutations of the human RAS genes were linked to tumorigenesis.
HRAS
Genetic analyses found that RAS is one of the most deregulated oncogenes in human cancers. In this review, we
KRAS
summarize the pioneering works which allowed the discovery of RAS oncogenes, the finding of frequent mu-
NRAS
Cancer tations of RAS in various human cancers, the role of these mutations in tumorigenesis and mutation-activated
Personalized medicine signaling networks. We further describe the importance of RAS mutations in personalized or precision medicine
particularly in molecular targeted therapy, as well as their use as diagnostic and prognostic markers as ther-
apeutic determinants in human cancers.

1. Introduction
Ras proteins are small GTPases which comprise three RAS proto-
oncogenes such as HRAS, KRAS, and NRAS in Homo sapiens (humans),
and they are orthologues of Hras (Harvey rat sarcoma virus oncogene),
Kras (Kirsten rat sarcoma virus oncogene) and Nras (Neuroblastoma ras
oncogene) in mice. The Ras genes are involved in the modulation of
various key cellular signaling pathways and regulate vital cellular
functions such as differentiation, cell proliferation, growth, cell cycle,
and motility. In this article, Ras gene nomenclature is used as re-
commended by HGNC (HUGO Gene Nomenclature Committee) and as
coined previously [1]. We use accordingly here, as, ras for viral origin,
RAS for the human origin and Ras for both viral and human origins. The
RAS is implicated in many human diseases including cancer and it has
been set as a "hallmark cancer gene" as it is the most mutated oncogene
in human cancers [1–4]. The term "ras" was originally derived from the
rat sarcoma virus and it denotes a common set of genes of rat-derived
murine sarcoma retroviruses responsible for transformation and tu-
morigenesis [5].
Ras encoded proteins belongs to the family of small GTPases. The
Ras family of small GTPases genes consists of approximately 36 mem-
bers, which encode about 39 Ras proteins. The molecular weight of
these encoded Ras proteins ranges from 20 to 29 kDa. It has been shown
that Rap (Ras-associated protein-1), Rit (Ras-like without CAAX 1), Ral
(Ras-like proto-oncogene), Rras2 (Ras-related 2) and Ras are belonging
to the same branch in the Ras family tree. Each member of the
branching Ras family has more than one isoform. The main branch of
the Ras family tree consists of several members such as Rap1A, Rap1B,
Rap2A, Rap2B, and Rap2C (Ras-associated protein-1A, B and 2A-2C,
member of Ras oncogene family), Rit1 (Ras-like without CAAX 1), Rit2
(Ras-like without CAAX 2), RalA isoform 1 (Ras-like proto-oncogene A
isoform 1), RalA isoform 2 (Ras-like proto-oncogene A isoform 2), RalB
(Ras-like proto-oncogene B), Eras (ES cell-expressed Ras), Hras isoform
1 (HRas proto-oncogene isoform 1), Hras isoform 2 (HRas proto-onco-
gene isoform 2), Kras2A (KRas proto-oncogene isoform 2A), Kras2B
(KRas proto-oncogene isoform 2B), Nras (NRas proto-oncogene), Mras
(Muscle Ras oncogene homolog), Rras (Ras-related) and Rras2 (Ras-
related 2) [6,7]. Though there are more than 39 various proteins, many
of them were not very well characterized except for HRAS, KRAS, and
NRAS. In normal cells, most of the characterized ras family proteins
have been found to be involved in the signaling pathways which reg-
ulate important cellular functions. When deregulated, these proteins
induce abnormal cellular proliferation, growth, survival, cytoskeletal
rearrangements, adhesion, motility, and differentiation resulting in
malignant tumor formation [8]. Here, we summarize the origin and
evolution of Ras research in its golden epochs, deregulated RAS genes in

Corresponding author. Present address: Department of Molecular Oncology, King Faisal Specialist Hospital & Research Centre, PO Box 3354, Research Center
⁎

(MBC 03), Riyadh, 11211, Saudi Arabia.

⁎⁎
Corresponding author.
E-mail addresses: akmurugan@gmail.com (A.K. Murugan), ntutid.eme@tmd.ac.jp (N. Tsuchida).

https://doi.org/10.1016/j.semcancer.2019.06.007
Received 28 November 2018; Received in revised form 13 May 2019; Accepted 7 June 2019
1044-579X/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Avaniyapuram Kannan Murugan, Michele Grieco and Nobuo Tsuchida, Seminars in Cancer Biology,
https://doi.org/10.1016/j.semcancer.2019.06.007
A.K. Murugan, et al.
Fig. 1. Timeline: milestones in the Ras research.
Timeline shows the important key discoveries of Ras oncogenes in the field of cancer research. The related reference numbers [10,12,13,16,17,19,25,28,42–44,
49,50–54,56,58,69,71,74,78,83,89,96,102,143–146] are mentioned within the bracket at the end of the sentences.
human cancer, mutated RAS-induced signaling, RAS mutations and
their importance in precision medicine particularly in molecular tar-
geted therapy, as diagnostic and prognostic markers in human cancers
is discussed.
2. Discovery of ras oncogenes: at the bench
The pioneering work that led to the discovery of the ras oncogenes
was started almost half a century ago and boomed in the early 1980s
and still, ras-related research findings are hot pop-ups in the field of
molecular and cellular biology. Moreover, cancer biology is expecting
every day yet other findings on the role of RAS genes in human cancer.
Retrovirus researches eventually lead to the finding of ras oncogenes.
As illustrated in Fig. 1, the discovery of the Rous sarcoma virus (RSV)
can be traced back to approximately 100 years ago. In 1911, Peyton
Rous discovered the RSV virus, when he was working at The Rockefeller
University in New York. He injected cell-free extract of a chicken tumor
into healthy Plymouth Rock chickens, the extract induced a tumor of
the connective tissue (a sarcoma) [9,10]. Therefore, it was termed as
Rous sarcoma virus (RSV). The RSV is a retrovirus, causes sarcoma in
chickens and is the first oncovirus to have been described. Rous was
awarded the Nobel Prize for his discovery in 1966, almost 55 years after
his discovery of RSV when Rous was 87 years old [11].
In 1964, Jennifer Harvey observed sarcomas in new-born rodents
which were inoculated a preparation of a murine leukemia virus, taken
from a leukemic rat [12]. Harvey's findings initiated ras discoveries,
subsequently; three additional studies found that three retroviruses also
carry ras oncogenes. Firstly, in 1967, Kirsten demonstrated that he
could obtain two serial passages of MSV (moloney sarcoma virus) cell-
free extract (later it was called Kirsten-MSV) which he was able to
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
transfer to newborn Wister-Furth rats [13]. Secondly, Peters et al iso-
lated BALB-MSV from a leukemic mouse in 1974 [14]. Thirdly, in 1978,
Rasheed et al, isolated stable rat sarcoma virus from cultured rat cells
[15]. Further, molecular cloning and characterization of Ha-MSV were
accomplished by Gordon L. Hager in 1979 [16]. Most of the sequences
responsible for the oncogenic properties of these acute transforming
viruses were molecularly defined in the early 1980s.
Molecular DNA clones of Ha-MSV and Ki-MSV genomes were iso-
lated and characterized by Ed Scolinick and his colleagues and Nobuo
Tsuchida's laboratory, respectively [16–18]. In 1981, Ed Scolnick and
his associates including Nobuo Tsuchida established that the Harvey
(Ha-MSV) and Kirsten (Ki-MSV) strains were recombinant viruses that
carry sequences derived from the rat 30S RNA genome [19]. KiMSV
clone 4 had been presented in a Cold Spring Harbor Laboratory (CSHL)
meeting of RNA tumor viruses (abstract, page 35 in 1980) much before
the publication of Ki-MSV cloning paper of clone 4, in 1981 [17]. Much
earlier to this publication, sub-clones of Ki-MSV clone 4 and Hi-MSV
clone H-1 were constructed and compared thus allowing to estimate the
area of the p21 transforming genes, then called src. Harvey and Kirstein
murine sarcoma viruses originated from divergent members of a family
of normal vertebrate genes (Hras in Ha-MSV and Kras in Ki-MSV) [19].
Among sub-clones, KBE2 and HiHi3 for Kras, and HB-11, and BS-9 for
Hras were used in most laboratories except for Aaronson’s and Barba-
cid’s labs, where human RAS genes were cloned by themselves and
identified using v-bas (mouse Hras) probes.
The first report of human KRAS sequence was published in August
1983 [20,21], one year after Nobuo Tsuchida's publication of the viral
Kras oncogene sequence which had transforming activity, published in
September 1982 [18]. Thus, the sequence of Kras in Tsuchida's paper is
the first Kras sequence to be reported. Robert Weinberg's laboratory
A.K. Murugan, et al. Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Table 1
Somatic mutation of the RAS oncogenes in human cancers.
S. No HRAS mutations (%)* KRAS mutations (%)* NRAS mutations (%)*
Primary tumor tissues Primary tumor tissues Primary tumor tissues

1 Salivary gland 24/164 (15%) Pancreas 3247/5656 (57%) Skin 918/5368 (17%)
2 Cervix 23/264 (9%) Large intestine 13177/38080 (35%) Hemato. & lymphoid 929/8882 (10%)
3 Upper aero-digestive tract 110/1202 (9%) Biliary tract 491/1766 (28%) Thyroid 330/4755 (7%)
4 Urinary tract 165/1765 (9%) Lung 2872/17472 (16.43%) Autonomic ganglia 6/102 (6%)
5 Penis 2/28 (7%) Small intestine 63/377 (17%) Adrenal gland 9/171 (5%)
6 Skin 134/2168 (6%) Endometrium 332/2268 (15%) Soft tissue 27/509 (5%)
7 Prostate 29/558 (5%) Ovary 436/3192 (14%) Large intestine 62/1731 (4%)
8 Soft tissue 37/740 (5%) Gastrointestinal tract 7/73 (10%) Upper aero-digestive 24/860 (3%)
9 Stomach 14/384 (4%) Prostate 94/1214 (8%) Liver 8/310 (3%)
10 Testis 5/130 (4%) Cervix 46/637 (7%) Ovary 5/191 (3%)
11 Thyroid 153/4231 (4%) Peritoneum 6/87 (7%) Testis 8/283 (3%)
12 Pituitary 10/300 (3%) Soft tissue 73/1100 (7%) Cervix 2/132 (2%)
13 Bone 3/199 (2%) Stomach 195/2814 (7%) Biliary tract 6/287 (2%)
14 Thymus 1/46 (2%) Liver 31/507 (6%) Pancreas 5/306 (2%)
15 Adrenal gland 1/136 (1%) Hemato. & lymphoid 310/6116 (5%) Stomach 5/215 (2%)
16 Breast 5/720 (1%) Genital tract 1/25 (4%) Urinary tract 10/873 (1%)
17 Endometrium 3/291 (1%) Testis 17/432 (4%) Breast 7/508 (1%)
18 Esophagus 2/161(1%) Penis 1/28 (4%) Prostate 8/588 (1%)
19 Autonomic ganglia 0/63 (0%) Urinary tract 47/1099 (4%) Central nervous system 8/1069 (1%)
20 Biliary tract 0/153 (0%) Eye 4/90 (4%) Endometrium 2/314 (1%)
21 Central nervous system 0/964 (0%) Autonomic ganglia 2/63 (3%) Lung 28/3084 (1%)
22 Eye 0/33 (0%) Breast 24/789 (3%) Eye 1/106 (1%)
23 Gastrointestinal tract 0/1 (0%) Esophagus 13/375 (3%) Fallopian tube 0/2 (0%)
24 Hematopoietic& lymphoid 8/3076 (0.2%) Salivary gland 6/173 (3%) Bone 0/207(0%)
25 Kidney 1/273 (0.3%) Upper aero-digestive 54/1673 (3%) Gastrointestinal tract 0/2 (0%)
26 Large intestine 2/717 (0%) Skin 39/1532 (3%) Kidney 2/435 (0%)
27 Liver 1/271 (0%) Thyroid 116/5259 (2%) Meninges 0/77 (0%)
28 Lung 9/2094 (0%) Thymus 4/186 (2%) Esophagus 0/161 (0%)
29 Meninges 0/75 (0%) Bone 2/252 (1%) Parathyroid 0/100 (0%)
30 Ovary 0/152 (0%) Kidney 4/704 (1%) Penis 0/28 (0%)
31 Parathyroid 0/100 (0%) Central nervous system 9/1175 (1%) Pituitary 0/300 (0%)
32 Peritoneum 0/3 (0%) Fallopian tube 0/3 (0%) Placenta 0/2 (0%)
33 Placenta 0/2 (0%) Adrenal gland 1/211 (0%) Pleura 0/30 (0%)
34 Pleura 0/19 (0%) Meninges 0/62 (0%) Salivary gland 0/48 (0%)
35 Pancreas 0/279 (0%) Parathyroid 0/100 (0%) Small intestine 0/5 (0%)
36 Small intestine 0/5 (0%) Pituitary 0/300 (0%) Thymus 0/46 (0%)
37 Vulva 0/16 (0%) Placenta 0/10 (0%) Vagina 0/1 (0%)
37 – – Pleura 0/45 (0%) Vulva 0/16 (0%)
39 – – Vagina 0/2 (0%) – –
40 – – Vulva 0/16 (0%) – –
742/21,783 (3.41%) 21,724/95,963 (23%) 2410/32,104 (7.51%)

* Mutated tumor Samples/Total number of tumor sample analyzed (%); Hemato., hematopoietic tumors. The data were derived from COSMIC (Catalogue Of
Somatic Mutation in Cancer) database.

was the first to report the transformation of NIH-3T3 cells with genomic
DNA of chemically transformed mouse cells [22] and subsequently,
they also showed the presence of an Alu repetitive sequence in the NIH-
3T3 cells which had been transformed with DNA isolated from human
tumor cell lines [23]. Alu repetitive sequences are human-specific and
they allowed the identification of human gene fragments inserted by
transfection in the mouse genome. In late 1981, Wigler and his group
reported the presence of Alu-containing DNA fragments in foci which
were obtained transfecting DNA from human tumor cell lines in mouse
cells [24]. In 1982, Weinberg and his group collaborating with Ed
Scolnick described a point mutation of human HRAS in bladder carci-
noma cells [25]. The Levinson's group used the KBE2 HiHi3 probe for
cloning human KRAS [26], while the Wigler's group used an Alu-spe-
cific probe in transfected-NIH-3T3 cells [27]. However, the uses of viral
Kras sub-clones were essential from the point that some of EcoRI
fragments of human KRAS DNA do not contain Alu sequence. Le-
vingson's group used HiHi3 to clone human KRAS sequence [28]. In
Early 1982, human transforming genes were cloned from T24 and EJ
bladder carcinoma cell lines and reported independently by Weinberg,
Wigler and Barbacid laboratories [29–31].
The molecular nature of these human oncogenes was resolved when
Channing Der used retroviral probes to hybridize DNA isolated from
NIH-3T3 cells which had been transformed with various human tumor
DNAs [32]. Wigler's lab and Barbacid's lab in collaboration with Stuart
Aaronson's lab, independently reported similar findings when they used
probes specific for v-Hras/v-Bas and v-Kras to hybridize the DNA iso-
lated from cells which were transformed by EJ bladder (EJ) and lung
(LX-1) carcinoma cell line DNA, respectively [33,34]. Interestingly four
years later, Weinberg’s group found that the initial NIH-3T3 transfor-
mants reported by them were carrying Kras oncogene [35].
The functional differences caused by single nucleotide were found
after cloning the normal and oncogenic allele of human HRAS in 1983
[25,27,36]. Unlike other oncogenes which were established as onco-
genes in the "post-ras-genomic era", the ras genes did not take a cake-
walk to the "cancer-podium", contrary, they took many criticisms when
they were considered as oncogenes due to a single point mutation in the
oncogenic allele. The question was raised because of two reasons:
firstly, as many mutations as arise in somatic cells every day, however
no tumor is formed until old age [37,38]; secondly, ras genes, unlike
their retroviral-counterparts oncogenes, were not able to transform
primary cells unless immortalized cells such as NIH-3T3 cells [39–41].
Moreover, the c-terminus of the RAS molecule was shown to be re-
quired for anchoring to the cell membrane and transformation [42].
Eventually, a study effectively showed that the cooperation of a second
immortalizing oncogene such as myc or adenovirus E1A was required to
induce tumor growth in nude mice [39,41]. Besides, a new human

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A.K. Murugan, et al.
transforming gene was identified in 1983. This gene was termed as
NRAS, which later became the third members of the Ras gene family,
and it was also shown to be mutated in human cancers [21,43]. How-
ever, the production of an NRAS deficient mouse showed that it is not
important for the growth and development but essential for immune
response and T-cell function [44].
The first RAS mutation in human cancer tissue was identified in
1984 when the KRAS gene was found to be mutated in the lung tumor
but not in the blood cells of the patient [34]. In the later years, mutation
analyses found a frequent mutation of KRAS gene in the colon [45,46],
lung [47] and pancreatic [48] cancers and it has been experimentally
demonstrated that the KRAS could cause early-onset lung cancer in
mice if somatically activated [49]. The Kras but not Hras and Nras were
shown to be important for the embryonic development in mouse [50].
Further, RAS was implicated in tumor maintenance [51]. In 1988, the
3D structure of human HRAS was determined [52], and the nucleotide
exchange-mediated activation of RAS and farnesyl transferase activity
was determined [53,54]. Further, studies were expanded to other iso-
forms of RAS, such as HRAS, and NRAS. In the following years, it has
been found that RAS is one of the frequently mutated oncogenes in
human cancers [2,3,7,45]. In 1973, many studies were conducted on
the nucleic acid sequences of Kirsten-MSV and a model was proposed
for the formation of a mammalian RNA-containing sarcoma virus by
Scolnick et al and sarcoma virus-related RNA sequences were found in
normal rat cells by Tsuchida et al in 1974 [55–57]. Viral Kras and Hras
genes were found to be originated from normal cellular genes. Although
after finding of ras genes opened the first door to the field of cancer
research in human cancers, which could never be accomplished from
src or mos researches since the src and mos genes mutations were never
been found in human cancers or it could be a rare event if there is. In
1976, Michael Bishop and Harold E. Varmus in collaboration with Peter
K. Vogt discovered that the DNA specific to the transforming avian
sarcoma virus was present in normal avian DNA [58]. This research
paved the way for the Nobel Prize in physiology or medicine in 1989.
The Nobel Prize was jointly awarded to J. Michael Bishop and Harold E.
Varmus for the discovery of the cellular origin of retroviral oncogenes
(Fig. 1). However, human src sequence has not been found as trans-
forming oncogene. Thus transforming RAS is the first human oncogene.
The history of human oncogenes and ras contribution to cancer re-
search was recently reviewed by Tsuchida et al [1].
3. Identification of frequent RAS mutations in human cancers
Finding of mutation in lung tumor ignited the researchers to search
for somatic mutations in other tumor types. Almost 35 years passed
since the first mutation was found in 1984 [34]. Overall, RAS is mu-
tated approximately 30% of human cancers. To date, in HRAS, overall,
741 mutations have been found in 21,793 various tumor samples ac-
counting for 3.41% mutations. The prevalence of the mutation varies in
different types of primary tissues. For example, the highest incidence of
HRAS mutation has been found in salivary gland tumors (15%). This
HRAS mutational incidence has truly been reflected also in the in-
dividual studies from various cohorts in oral squamous cell carcinomas
(OSCC) of head and neck [59–61] and a relatively high incidence of
mutations have also been found in cervical, upper aero-digestive tract
and urinary tract (9%) (Table1).
In the KRAS, 21,724 mutations have been found from 95,963
samples analyzed and accounting for 23% of mutations in human
cancers. The KRAS is found to be the most mutated oncogene in human
cancers. The highest incidence of mutations has been found in pan-
creatic cancer (57%). Relatively, a high incidence of mutations has also
been found in malignancies of the large intestine (35%), biliary tract
(28%), small intestine (17%), lung (16%), endometrium (15%) and
ovary (14%) as listed in Table 1. In the NRAS, 2410 mutations have
been identified in 32,104 tumors overall accounting for 7.51% of mu-
tations in various primary tumor tissues. A very high incidence of NRAS
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
mutations has been identified in malignant melanomas (17%). A rela-
tively high incidence of the mutation has also been found in malig-
nancies of hematopoietic and lymphoid tissue and thyroid cancer re-
presenting somatic mutation of NRAS 10% and 7%, respectively.
Though the mutations of NRAS were also often detected in other tu-
mors, their prevalence is relatively low, accounting for 0–5% (Table 1).
4. Roles of RAS mutations in human cancers
The RAS mutations play an important role both in upstream and
downstream of the onset of the disease, cancer. The upstream of the
onset disease shows the course of developing the disease that con-
stitutes the acquiring the RAS mutations, developing the disease and
exhibiting the first or initial phenotype of the disease with primary
symptoms. In a more simplified way the carcinogenesis process.
Further, the downstream of the onset covers the possible outcome of the
disease and the frequency with which the disease could be expected in
the future to occur. In a simplified way, the downstream of the cancer
onset is the prognosis, a prediction of the course of a disease following
the disease onset.
4.1. Oncogenic mutations of RAS genes
RAS oncogenes are mutated with different prevalence in human
cancers (Table 1). No specific pattern can be identified and/or specu-
lated based on etiological and ethnic factors except some types of
cancers such as oral cancer, pancreatic cancer, and lung cancer. For
example, a relatively high incidence of HRAS mutations have been
identified in oral cancers obtained from patients who were exposed to
some nitrosamine containing carcinogens through the habit of tobacco
chewing, smoking and betel quid, etc. [60,61]. The possible role ex-
erted by these carcinogens has been proved in experimental models by
inducing HRAS single nucleotide point mutations in the tumor of a
mouse using nitrosomethylurea [62]. Many animal models have been
demonstrated to show that tobacco-derived compounds could induce
KRAS mutations and that these mutations have occurred at the begin-
ning of the tumorigenesis process [63]. Approximately, 7–8 fold in-
creased incidence of KRAS mutation has been observed in a pancreatic
cancer patient group with higher occupational exposure to organic
solvents [64]. Similarly, tobacco smoke-induced KRAS mutation has
been a well-known fact for the past 20 years [8,65].
Although the RAS mutations are widely distributed throughout the
RAS genes, some amino acid residues are predominantly found to be
mutated in cancer (Fig. 2). For example, three amino acid residues such
as G12, G13, and Q61 of all HRAS, KRAS, and NRAS are highly mutated
in human cancers (Figs. 2 & 3 ). These mutations prevent the intrinsic
and GAP catalyzed hydrolysis of GTP, thereby generating constantly
active ras molecule [66]. In a normal cell, equilibrium is tightly
maintained between the active and inactive state even though RAS
proteins have a minimal and measurable activity on their own [61].
This condition is strictly maintained by GAPs (GTPase activating pro-
teins) and GEFs (Guanine nucleotide exchange factors) in the normal
physiological condition of the cell. The GAPs such as (NF1 and
p120GAP) accelerate the GTP hydrolysis of ras and the antagonist GEFs
such as ras-GRFs (p140 RAS-GRF1 and p130 RAS-GRF2) and RAS-GRPs
(RASGRP1 and RASGRP 2) catalyze and weaken the GDP replacing with
GTP [67,68]. In the pathophysiological condition of a cell where RAS is
mutated, the equilibrium between the GTP and GDP-bound state is
impaired. The constitutively activated RAS molecule triggers the sig-
naling on the downstream effectors that result in hyperactivation of
cellular pathways which control important cellular functions such as
growth, cell proliferation, survival, cytoskeleton rearrangements, ad-
hesion, motility, and differentiation [66]. By this uncontrolled activa-
tion, the cell undergoes the neoplastic transformation that results in
cancer. RAS molecules and their various modulators in human cancer
were recently reviewed [2].
A.K. Murugan, et al. Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Fig. 2. Schematic diagrams of the RAS oncogenes showing mutations reported in various human cancers.
A. Schematic illustration of HRAS gene. The HRAS is located on chromosome 11p15.5 and contains 4 exons with intervening sequences. Shown are various oncogenic
somatic mutations of the HRAS gene identified in various human cancers. B. Schematic sketch of KRAS gene. The KRAS has two sub-types such as KRAS A and B. The
KRAS is located on chromosome 12p12.1 and contains 4 exons with intervening sequences. All the oncogenic somatic mutations of the KRAS gene are marked in the
illustration. C. Schematic picture of NRAS gene. The NRAS is located on chromosome 1p13.2 and contains 4 exons with intervening sequences. Various human cancer-
associated oncogenic somatic mutations of NRAS gene are marked here. The missense somatic mutations identified in RAS genes of various human cancers were
derived and analyzed from COSMIC (www.sanger.ac.uk/genetics/CGP/cosmic/).

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A.K. Murugan, et al. Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Fig. 3. Space-fill model 3D structure and differential interacting network of the RAS oncoproteins.
A. Figure shows the space-fill model of HRAS. B. The space-fill model of KRAS. C) It shows the space-fill model of NRAS. Shown are the space-fill diagrams of a native
modeled structure of HRAS, KRAS, and NRAS, respectively. The protein structure PDB ID of HRAS, KRAS and NRAS are 121 P, 5VP7, and 5UHV, respectively. Amino
acid residues of the frequently mutated codons in human cancer were traced and plotted in RAS protein structures and protein 3D molecules were visualized by NGL
(WebGL) viewer. D. An interacting network of HRAS. E. An interacting network of KRAS. F. An interacting network of NRAS. Interacting network for each member of the
RAS protein was built by the STRING version 10.5 setting with highest confidence interaction score (0.900) and kmeans clustering with a minimum 5 number of
clusters. The result of the network analysis was imported as high-resolution portable network graphic (PNG) file.

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A.K. Murugan, et al.
4.2. Mutant RAS-induced constitutive activation of vital signaling pathways
Constitutively activated RAS activates various downstream sig-
naling molecules such as PI3K (Phosphatidylinositol 3-kinase), RAF (A
family of three serine/threonine-specific protein kinase, an acronym for
Rapidly Accelerated Fibrosarcoma), Rin1 (Ras and Rab Interactor 1),
RASSF (Ras association domain family member), AF6 (Afadin protein),
PLCε (Phospholipase C-epsilon), RalGEF (Ras-like Guanine nucleotide
Exchange Factor), Tiam1 p190 (T-lymphoma invasion and metastasis 1
p190), PKCξ (Protein kinase C epsilon type), etc., which control many
cellular processes such as growth, cell proliferation, survival, cytoske-
leton rearrangements, adhesion, motility and differentiation including
tumorigenesis [2,3,7,8,66]. As illustrated in Fig. 4, receptor tyrosine
kinases signaling were shown to be linked to Ras signaling via SH2 and
SH3 domain-containing adapter GRB2 [69]. The RAF1 mediated MAPK
cascade and the PI3K/AKT signaling pathways are the two key down-
stream pathways of constitutively activated RAS signaling involved in
tumorigenesis. The Ser/Thr kinase RAF1 and its interaction with RAS
was found in 1993 by independently four groups and RAF1 was the first
mammalian RAS effector to be found [70–73]. Subsequent extensive
research found that the RAF1 signal through mitogen-activated protein
kinases (MAPK). RAS interaction with RAF1 kinase facilitates the
phosphorylation of two distinct serine residues of the dual specificity
protein kinases MEK1/2, which, in turn, activate by phosphorylating
threonine and tyrosine residues in the activation loop of the serine/
threonine-specific protein kinases ERK1/2 that results in constitutive
activation of the MAPK signaling pathway. In addition, it has been
shown that CRAF but not BRAF is required for KRAS (G12D)-mediated
initiation of lung cancer [74]. The activated MAPK cascade promotes
cell differentiation, growth and cell cycle. Subsequent remarkable stu-
dies on RAS-induced MAPK signaling found that this cascade is suffi-
cient and important for RAS-induced transformation of murine cell lines
[75–77].
In addition to RAS-induced RAF1 mediated MAPK cascade, the PI3K
signaling pathway is another important RAS-induced pathway [78].
PI3K is a heterodimeric lipid kinase which is composed of a catalytic
subunit (p110α) and a regulatory subunit (p85) [79]. The p110α con-
tains a RAS-binding domain (RBD) which enables p110α to interact
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
Fig. 4. Mechanisms of the Ras activation and
its major signaling pathways.
The active-inactive cycle of RAS is tightly
controlled by GAPs (GTPase-activating pro-
teins) and GEFs (guanine-nucleotide exchange
factors). Mutated RAS is constitutively acti-
vated as it is constantly bound to GTP. Upon
the RAS activation, the MAPK, and PI3K/AKT,
the two major downstream signaling pathways
are activated and these pathways are asso-
ciated with growth, differentiation, develop-
ment, proliferation and survival.
with the RAS-GTP bound form [80,81]. The association of PI3K activity
with RAS activation was reported by Lapetina and colleagues in 1991
[82]. Subsequently, three years later, in 1994, the direct interaction of
p110α of PI3K with the RAS was demonstrated by Julian Downward
and his group [78]. More specifically, binding of RAS to (p110α) PI3K
is necessary for RAS driven tumorigenesis [83]. Among the three RAS,
HRAS has been shown to be more efficient than KRAS in activating PI3K
[84]. RAS-mediated activation of PI3K generates abundantly the second
messenger PIP3 (phosphatidylinositol-3,4,5-triphosphate) from PIP2
(phosphatidylinositol-4,5-biphosphate). The tumor suppressor PTEN
antagonizes PI3K activity by dephosphorylating the PIP3 to PIP2
[80,81]. Generation of the second messenger, PIP3 recruits pleckstrin-
homology (PH) domain-containing proteins such as PDK1 and AKT/
PKB to the plasma membrane, and facilitates the activation and phos-
phorylation of AKT at threonine 308 by PDK1 [85]. AKT1 has been
known to be involved in cell proliferation and survival, AKT2 has been
linked to insulin-mediated metabolic process while AKT3 has been in-
volved in cell size and cell number, altogether proven to play an im-
portant role in tumorigenesis [86]. Activated AKT initiates protein
synthesis through a cascade of its effector proteins such as mTOR
(mammalian target of rapamycin) acting on to two critical downstream
targets, S6K (p70S6K) and 4EBP1 (eukaryotic initiation factor 4E-
binding protein 1). MTORC2 phosphorylates and thereby additionally
activates AKT at serine 473 [2,87]. AKT also regulates transcription-
induced phosphorylation-dependent degradation of FOXO1 (forkhead
box O transcription factor). Active PI3K also activates Rac [88].
Moreover, it has been found that RAS and RHO families of GTPases
were shown to directly regulate various isoforms of PI3K [89]. Re-
cently, noncoding RNAs (ncRNAs) were shown to act as regulators of
RAS oncogenes, these ncRNAs epigenetically modulate the expression
of RAS genes and loss of RAS-repressing ncRNAs results in upregulation
of the RAS oncogenes [90]. These RAS mutation-induced constitutive
activation of various vital signaling molecules collectively result in
uncontrolled cell proliferation and growth, cell survival and motility.
The ncRNAs-mediated epigenetic regulation also suggests an alter-
native and mutation-independent activation mechanism of RAS onco-
genes (Fig. 4).
A.K. Murugan, et al.
5. Role of RAS mutations in precision medicine for human cancer
Precision medicine can be defined as the tailoring of the therapeutic
strategies (developing effective, safe medications and doses) based on
genetic (genomics, transcriptomics, proteomics), environmental and
lifestyle factors. Pharmacogenomics becomes a part of the precision
medicine as it deals the effect of genes on a person’s response to par-
ticular drugs. Although "precision medicine" and "personalized medi-
cine overlap each other, National Research Council (NRC), USA ap-
proved and preferred the term "precision medicine" rather than the
term "personalized medicine" as the later one is relatively older and
could be misinterpreted nevertheless the two terms are still used in-
terchangeably by some people. RAS mutations could be used as an
important factor in the determination of the therapeutic option for
human cancer. RAS is one of the most sought therapeutic targets, di-
agnostic and prognostic markers in human cancers.
5.1. Targeted therapy for RAS mutation in human cancers
Developing an effective small molecule against the molecular target
is the initial phase of precision medicine. Direct inhibition of mutant
RAS has been attempted in various ways, however; it has been a big
challenge forever with unique difficulties in each methodology due to
multifaceted personalities of RAS oncoproteins. Principally RAS iso-
forms displayed their variable activation across the human cancers that
reflected the differential effects of RAS mutations [7,61,91]. Therefore,
to bypass these difficulties, the downstream signaling pathway of RAS
was targeted. Unlike other proteins, RAS signals through multiple
mediators and effectors [7]. This complexity raised the question "which
pathway should be targeted?” On the other hand, cancer is a multi-
factorial genetic disease, potentially more than a gene is required for
the tumor initiation, maintenance, and metastasis and it would be
practically not feasible to use a single inhibitor which could conquer the
tumorigenesis. Therefore, more than an inhibitor is necessitated to see a
clinically significant response in a setup/pathway where many genes
are deregulated. Besides, it is also not possible to use more than two
drugs/inhibitors as the drug itself could cause serious side-effects and
most of the cancer drugs targets invariably both the normal and cancer
cells. Therefore, minimal drug and clinically a maximal effect are
needed. This prompted the researchers to narrow down the number of
targets based on the direct target and closely signaling pathways which
are involved in proliferation, growth, tumorigenesis, survival, and anti-
apoptosis.
Well-characterized and the direct downstream of the RAS signaling
are the MAPK and PI3K signaling pathways [92] and MEK and AKT
inhibitors block these two pathways, respectively. Though the MAPK
pathway is preferentially activated by RAS, it has also been known to
interact with p110α, the catalytic subunit of PI3K and PI3K signaling is
firmly established under RAS [83]. Furthermore, in KRAS driven col-
orectal tumor, the PIK3CA mutation increase the resistance to EGFR
therapy [93]. Therefore, the PI3K pathway inhibition becomes neces-
sary. The use of these inhibitors may be an alternative, appropriate and
a promising strategy for targeting RAS-mediated signaling pathways in
human cancers because this therapeutic strategy not only inhibits RAS-
mediated signaling, but it paves also the way for inhibiting other ge-
netically altered upstream and downstream effectors of RAS. Moreover,
this strategy also inhibits many other cancer-associated activated pro-
tein molecules which trigger the growth signal through AKT and MEK.
This may have a synergistic effect on both PI3K/AKT pathway and RAS-
RAF-MEK-ERK cascade. The molecular targeted therapy (MTT) acting
on these two pathways should be taken into account at least in a subset
of patients with RAS mutations.
In the future, the precision medicine will depend mainly on the
genetic makeup of the patients as their genotype could drive the best-
targeted therapies. To this end, finding allele-specific inhibitors for RAS
mutants would predominately bring success to the precision medicine
8
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
from bench to bedside. Recently, this task has been achieved only for
the G12C KRAS mutated form developing a small molecule inhibitor
that actively bound covalently to cysteine and was shown to inhibit
oncogenic RAS [94,95]. As seen in Fig. 5, it has been shown that a RAS
inhibitor, ARS853 could selectively interact to the GDP-bound KRAS
and block the nucleotide exchange which is important for the produc-
tion of GTP-bound form by the mutated codon 12 [96]. Similarly,
acetylation-mediated inhibition of oncogenic activity has been shown
for the codon lysine 104 KRAS [97]. On the other hand, inhibition of
nucleotide exchange itself demonstrated as an alternative therapeutic
strategy, wherein a synthetic peptide has been shown to bind to the RAS
and inhibit the interaction of Son of Sevenless (SOS) that could result in
the prevention of GEF-induced nucleotide exchange [98]. These mole-
cular insights exploited the nucleotide exchange mechanisms-mediated
activation of RAS molecules that helps to lock the mutant RAS mole-
cules to the GDP-bound state as constantly inactive. Moreover, an al-
ternative mechanism has been suggested to explore, i.e., understanding
differential activation of various effector pathways and utilize them
using various inhibitors as mutant alleles are not alike each other [99].
Furthermore, Tipifarnib – a RAS pathway inhibitor has been approved
by USFDA for the treatment of acute leukemia, especially in elderly
cases. Moreover, some peptidomimetics and bi-substrate inhibitors are
under clinical trials (FTI 276, FTI 277, B956, B1086, L731, L735, L739,
L750, BMS-214662, and L778123) and they are likely to pave the way
for precision therapy in RAS-mutant positive cases [100]. Recently, it
has been shown that inhibition of galectin-3, a glycoprotein that binds
to integrin αvβ3 sensitizes the mutant KRAS-addicted tumors to therapy
and integrin αvβ3 might be used as a biomarker to identify mutant
KRAS-addicted tumors. Moreover, galectin-3 directly binds to the cell
surface receptor, named integrin αvβ3 that result in KRAS addiction via
acquiring various functions of KRAS in transformed cells and that
makes the galectin-3 as a lynchpin for KRAS-addicted tumors [101].
Recently, various small-molecule pan-RAS ligands have been synthe-
sized and a compound (named 3144) was shown to bind to RAS pro-
teins. The small molecule found to interact with the oncogenic KRAS
particularly with adjacent sites on the surface of KRAS. This pan-RAS
inhibiting molecule showed promising and metabolically stable anti-
cancer effect in the xenograft mouse models and pose an effective anti-
cancer therapeutic strategy for human cancers with RAS mutations
[102]. Although drugging RAS has initial failures, after great effort and
understanding the complexities of RAS now armed with new directions
with various technologies [103]. Recently, the NCI (National Cancer
Institute) has initiated a suite for next-generation anti-RAS drug dis-
covery [4]. This initiative gives a big hope that could develop new
armor which alters the undruggable RAS to druggable. This would help
in utilizing various pathway-specific inhibitors which are in the market
and clinical trials for the cancer patients bearing RAS mutants with
codon-specific pathway activation.
5.2. Inhibition of RAS molecules in human cancers and the mechanism of
resistance
Targeting RAS itself is a cumbersome matter as each mutant of these
RAS molecules exhibits differential phenotypes; moreover, the majority
of the cancer therapies fails as a result of resistance. It has been clearly
demonstrated that the inhibition of RAS oncogenes could cause a dra-
matic regression of various tumors in mouse models. Nevertheless,
emerging of tumor resistance as a result of various new RAS-in-
dependent clones was observed which were mostly shown to be driven
by the YAP1 gene [104]. Similarly, this phenomenon has been alter-
natively demonstrated showing that YAP1 gene overexpression could
rescue cancer cells from the depletion of KRAS molecules [105]. Cur-
rently, HRAS is inhibited using farnesyl transferase inhibitors which are
presently used in clinical trials and mechanism of resistance if any
would tend to emerge in the future. The major advantage of the RAS
inhibitors is that they could likely relieve on the feedback loop in the
A.K. Murugan, et al.
Fig. 5. Trapping mechanisms of the KRAS G12C allele-specific inhibitor.
A RAS inhibitor, ARS853 selectively interact to the GDP-bound KRAS codon
G12C allele and block the nucleotide exchange which is important for the
production of GTP-bound form by the mutated codon 12 whereby lock the
mutant KRAS molecules to the GDP-bound state as constantly inactive.
upstream growth factor receptors. Although oncogenic mutations of
RAS, BRAF, and MEK genes are mutually exclusive in this MAPK
pathway, combination therapy may be necessary if RAS inhibitors are
used in clinics to overcome a concomitant mutation of BRAF/MEK.
Recently, it has been demonstrated that Kras G12D “knockin’’ mutant-
generated primary murine acute myeloid leukemia (AML) responded to
MEK inhibitor PD0325901 (PD901) and showed intrinsic drug re-
sistance at relapse. On the other hand, they found that the resistance
cases were sensitive to MEK inhibition when the wild-type KRAS is lost.
This was also reflected in the colon cancer cell lines when the WT KRAS
was changed to a mutant allele that caused the heterozygous mutant-
bearing cells sensitive to treatment while the KRAS mutants were al-
ready highly sensitive to MAPK inhibition in HCT116 cells. They also
showed that in advanced cancer cases, 642 of 1168 (55%) patients with
KRAS mutations showed allelic imbalance suggesting that the genetic
changes at KRAS locus are common in human cancer and they are likely
to be dependent (sensitive) on MEK inhibition [106].
5.3. The utility of RAS mutations as diagnostic markers in human cancers
The currently available diagnostic tests mainly and mostly show a
lack of sensitivity and specificity to identify the markers for early stages
of cancer regardless of molecular, metabolomics, invasive, non-invasive
nature of the test. Many studies have been carried out to determine the
diagnostic utility of KRAS mutations but the results were highly het-
erogeneous and contradictory. Pancreatic cancer is one of the most
lethal human cancers with less than 3% overall 5-year survival rate and
requires more clinically meaningful tests for early diagnosis. KRAS is
the most mutated oncogene in pancreatic cancer. Various methods have
been used to detect KRAS mutations in cytological tissues as a mole-
cular marker for the diagnosis of pancreatic cancer [107]. It has been
reported that KRAS mutations were mostly detected in cells derived
from invasive methods such as fine needle aspiration (FNA), endoscopic
retrograde cholangiopancreatography (ERCP), and surgery. When the
first two (FNA and ERCP) were compared with surgery, the pooled
sensitivity was 77% versus 54% and the specificity was 88% and the
diagnostic odds ratio (DOR) was 20.2 versus 7.52, respectively. Further,
the study suggested that diagnostic accuracy was significantly im-
proved when two methods were combined. A mouse model with age-
matched wild-type mice compared to p48-Cre/LSL-KrasG12D showed
that malignant progression of pancreatic ductal adenocarcinoma
(PDAC) is analogous to the human disease stages via early and late
9
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
pancreatic intra-epithelial neoplasia (PanIN) lesions. Accuracy rates of
distinguishing mice with early or late stage lesions versus their re-
spective controls were 82.1% and 81.5%, respectively [108]. Effective
management of pancreatic intraductal papillary mucinous neoplasms
(IPMN) and mucinous cystic neoplasms (MCN) are exclusively based on
distinguishing accurately the mucinous cyst from other pancreatic
cystic lesions. In fact, a recent preoperative testing KRAS in the diag-
nosis of pancreatic mucinous cysts showed KRAS mutations in 28 (68%)
IPMNs, 7 (78%) IPMNs with adenocarcinoma, and 1 (6%) MCN. KRAS
showed 100% specificity and 65% sensitivity for mucinous differ-
entiation. In the case of IPMNs, KRAS had 98% specificity and 84%
sensitivity [109]. In addition, the integration of KRAS testing in 618
pancreatic cysts showed a specificity of 100%, but a sensitivity of 54%
for mucinous differentiation [110]. The KRAS is frequently mutated in
colon cancer next to pancreatic cancer.
A study using the chip-based digital PCR for detection of circulating
tumor DNA in metastatic colon cancer revealed the detection of ctDNA
(circulating tumor DNA) in 11/16 cases (68.75%) with KRAS-mutated
tumors. It was consistent with the detection rate of the chip-based dPCR
(96%) and KRAS mutation was detected in 18 wild-type tumor cases
with a sensitivity and specificity of 69% and 100%, respectively [111].
Moreover, the fecal immunochemical test (FIT) has also been used as
one of the diagnostic tests to detect the KRAS mutation for diagnosis of
colon cancer [112]. Although RAS mutations incidence is relatively low
in bladder cancer, RAS is used as a potential diagnostic marker in urine
samples especially, the HRAS along with the TERT, FGFR3, and PIK3CA.
The concomitant presence of these gene mutations in urine samples was
not often revealed [113].
In the thyroid, nodules are evaluated by fine-needle aspiration
(FNA) cytology and 20–30% of FNAs often shows indeterminate cy-
tology that challenges the relevant management options [114]. Mostly,
follicular or oncocytic (FN)/suspicious follicular or oncocytic neoplasia
(SFN) is the common indeterminate diagnosis that poses the cancer risk
of ∼15–30%. A ThyroSeq v2 next-generation sequencing assay-based
approach for the diagnosis of cancer in thyroid nodules including FN/
SFN identified NRAS followed by KRAS as the most commonly mutated
oncogenes in the nodules. This assay showed 90% sensitivity and 93%
specificity with 92% accuracy and the positive and negative predictive
value was 83% and 96%, respectively [115]. A recent study found 17 of
362 nodules positive for RAS mutations out of which 8 (8/17, 47%)
proved to be malignant after surgery and also showed that the RAS-
positive nodules had an indolent behavior for many years as no extra-
thyroidal extension, metastases, or lymphovascular invasion with low
risk [116].
5.4. Use of RAS mutations as prognostic markers in human cancers
RAS is one of the most mutated oncogenes in human cancers. EGFR
is one of the other most genetically altered oncogenes in human cancers
and it is an upstream effector of RAS signaling. The EGFR is ther-
apeutically targeted using anti-EGFR antibody-mediated therapy,
especially in lung cancer and colon cancer. Recently, mutation status of
KRAS is determined before the EGFR-targeted therapy in human can-
cers, especially in lung and colon cancers [117,118]. Furthermore, the
determination of the presence or absence of the KRAS mutation has
been considered as an important key factor to predict prognosis and/or
the treatment method in these cancers [117,118]. Because of the pre-
sence of a KRAS mutation is observed as an adverse prognostic factor
[119] and also a predictor of failure to benefit from EGFR-targeted
therapy [120–122]. Apart from the lack of response, it has been
documented that KRAS mutations worsen the response when EGFR-
targeted therapy was added to their standard chemotherapy [121].
Approximately, 40% of colorectal cancers harbor with KRAS mu-
tations [123], among them, 90% of mutations are detected in the codon
12 and 13 while the remaining ones occur in codon 61 and 146
[124,125]. It has been predicted that mutation in the codon 12, 13 and
A.K. Murugan, et al.
61 may show a lack of response to cetuximab while codon 146 muta-
tions did not affect cetuximab efficacy [125]. In contrast, EGFR ex-
pression in colon cancer failed to demonstrate predictive value for anti-
EGFR monoclonal antibody therapy. The intensity of EGFR staining by
IHC analysis showed no correlation with the response rate to cetuximab
and panitumumab [126,127]. Therefore, KRAS mutation in human
cancer is an established predictor of response to EGFR-targeted therapy.
As it is well evidenced, obtaining a comprehensive mutational profile is
clinically highly necessary and this may help in treatment selection in a
molecular targeted therapy.
5.5. RAS mutations alter the outcome of the disease
Although all RAS genes are mutated at various extents in human
cancers, the KRAS mutations show the most frequent mutations and
influence the clinical outcome of the KRAS-associated tumors to the
worst condition. KRAS mutations are associated with clinical outcomes,
such as inferior survival in colon cancer patients [128,129] and me-
tastasis and invasion in pancreatic cancer [130]. The highest incidences
of codons 12 and 13 mutations have been detected in colorectal cancer
with liver metastasis (CRLM) and these mutations (G12V/C/S) were
correlated with poor overall survival [131]. In a mouse system, PI3K
activity had been reported to be essential both for the initiation and the
maintenance of tumors with the KRASG12D genotype and this activity
requires KRASG12D-PIK3CA (p110α) binding [3,83]. In addition, RAS
interaction with PIK3CA (p110α) is required for maintenance of lung
tumor and tumor-induced angiogenesis [132, 133]. In human carcino-
genesis, the mutated PIK3CA was demonstrated to be associated with
invasion [134]. Meanwhile, mCRC (metastatic colorectal cancer) pa-
tients with brain metastasis predominantly had the KRASG12D geno-
type rather than the other genotypes of codon 12 [135]. The
KRASG12D in NSCLC cell lines has more preferential signaling to the
PI3K pathway than to the Raf pathway [136]. It was reported that the
same mutant was a stronger stimulant of the Raf-ERK pathway than of
the PI3K-AKT pathway in lymphoid neoplasia [137]. Therefore, further
studies are needed to elucidate the detailed mechanisms of signaling to
downstream effectors, including differences in tumor cell types, KRAS
genotypes (amino acid substitutions), and isotypes (KRAS4A/KRAS4B).
It has been reported that KRAS4A mRNA was widely expressed in ex-
amined human cancer cell lines, though KRAS4B mRNA was present at
higher levels, but, amounts approximately equal in most cases of 17
fresh human colorectal tumors [138].
6. KRAS: a prime focus
Overall KRAS oncogene is the most frequently mutated (∼23%)
oncogene in human cancers compared with the other counter RAS
subtypes, HRAS (3.4%) and NRAS (7.5%). Moreover, KRAS is specifi-
cally deregulated in the most aggressive human cancers such as pan-
creatic (∼57%), large intestine/colon (∼35%), biliary tract (˜∼28),
lung (∼17%) suggesting an important role of KRAS in these carcino-
geneses. This observation paved way for PRIME and CRYSTAL, two
large-scale clinical trials. The prime idea was to incorporate anti-EGFR
mAbs as first-line chemotherapy for metastatic CRC patients however,
this therapy exhibited clear survival benefit only for cases with wt-
KRAS and NRAS. Moreover, this therapy showed worse/no benefit on
overall survival for cases bearing mutant KRAS/NRAS. Therefore, cur-
rently, cetuximab and panitumumab-mediated treatments are re-
commended only for the patients bearing wt-RAS suggesting that
evolving a KRAS-dependent precision medicine for mCRC [139]. The
KRAS codon 12 mutation subtypes seem to be different in biologic ac-
tivities while KRAS of G12D mutation is associated with poor prognosis.
Recent findings showed that the KRAS mutations have a differential
role even in a specific tumor type. The KRAS G12D mutants showed
poorer median survival when compared with Non-G12D KRAS mutants
and they mostly represented with advanced tumor stage in ampullary
10
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
cancer and the KRAS G12D mutant could be a poor prognostic marker
in this cancer [140]. Similarly, non–small cell lung cancer (NSCLC)
patients with KRAS-G12C/G12 V exhibited worse progression-free sur-
vival compared with the patients bearing other KRAS mutations/wild-
type. Interestingly, NSCLC cell lines with KRAS-G12C/G12 V mutants
triggered the Ral signaling and abolished the growth factor dependence
for AKT activation. On the other hand, KRAS-G12D mutants activated
both the PI3K/AKT and MAPK signaling. Molecular modeling on these
mutants revealed that different confirmations resulted from various
mutants likely to have differential binding efficiency with the down-
stream signaling members which transduce heterogeneous mutant-
specific signaling [136]. These data suggest that KRAS mutants have
differential oncogenic signaling and patient outcome. Moreover, the
differential effect of KRAS mutants implies that therapeutic strategies to
be catered to the tumors based on the status of amino acid-specific
KRAS mutation. Recently, KRAS-specific inhibitors (Deltarasin & Sal-
irasib) have been synthesized and clinically evaluated [100]. NCI in-
itiative on drugging RAS may bring hope for patients with RAS muta-
tions particularly for KRAS as this gene is predominantly implicated in
human cancers especially in aggressive phenotypes such as pancreatic,
colon and lung cancer, and the KRAS seems to be much more important,
followed by NRAS and HRAS [4,141]. Nonetheless, biochemistry has
been relatively advanced with HRAS but that of KRAS was slightly
delayed. Recently, it has been demonstrated that cancers with KRAS
mutations could be targeted with a covalent G12C-specific small mo-
lecule inhibitor [142]. Further, a recent study traced the evolutionary
routes of KRAS dosage variation and their role in cancer phenotypic
difference, early tumorigenesis, and metastasis in pancreatic cancer
[143]. This uncovers the truth behind the differential cancer pheno-
types for a specific codon of KRAS.
7. Conclusion and future prospects
The RAS is one of the genetically most deregulated oncogenes in
human cancers. HRAS, KRAS, and NRAS are mutated with the highest
frequency in salivary gland (15%), pancreatic (57%) and skin cancer
(17%), respectively. More than three decades of RAS research have
unraveled the important role played by RAS genes in human cancers.
Detection and exploitation of RAS mutations for molecular targeted
therapy and as a diagnostic and prognostic marker in precision medi-
cine is an indicator of the best therapeutic approach. In future, a po-
tential and effective RASopathy would exclusively be depending on
designing various potent inhibitors which could significantly inhibit the
unique, multifaceted and differential personalities of various alleles of
mutant RAS molecules and their closely cooperating partners with least
side effects.
Acknowledgements
This work was partly supported by Grants-in-Aid from The Ministry
of Education, Culture, Sports, Science and Technology (MEXT), Japan
[Grant #06044072], Japan Society for the Promotion of Sciences
[Grant #257132] and National Institute of Health (NIH), U.S.A. [Grant
#CA22701] to N. Tsuchida as the P.I. We (A.K. Murugan and N.
Tsuchida) thank Government of Japan for awarding “Monbukagakusho:
MEXT Scholarship" to A.K. Murugan. We apologize that we could not
cite some of the remarkable work of our colleagues mainly due to space
limitations.
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