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EDITED BY

GEORGIA KONSTANTINIDOU



University of Bern, Switzerland

REVIEWED BY

LU SHAOYONG



Shanghai Jiao Tong University, China

ROBERTO GIOVANNONI



University of Pisa, Italy

The editor and reviewer's affiliations are the latest provided on their Loop
research profiles and may not reflect their situation at the time of review.

TABLE OF CONTENTS

 * * Abstract
   * Introduction
   * RAS Signaling Cascade and Regulation
   * Role of RAS Mutations in Different Cancer Types
   * RAS and Metabolism
   * RAS in Cancer Metastasis
   * RAS and the Immune System
   * Therapeutic Targeting of RAS
   * Conclusions
   * Author Contributions
   * Conflict of Interest
   * Acknowledgments
   * References


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REVIEW ARTICLE

Front. Oncol., 24 September 2019
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https://doi.org/10.3389/fonc.2019.00965

THIS ARTICLE IS PART OF THE RESEARCH TOPIC

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RAS: STRIKING AT THE CORE OF THE ONCOGENIC CIRCUITRY

Ryan C. Gimple1,2 and Xiuxing Wang3*
 * 1Division of Regenerative Medicine, Department of Medicine, University of
   California, San Diego, San Diego, CA, United States
 * 2Department of Pathology, Case Western University, Cleveland, OH, United
   States
 * 3Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical
   University, Nanjing, China

Cancer is a devastating disease process that touches the lives of millions
worldwide. Despite advances in our understanding of the genomic architecture of
cancers and the mechanisms that underlie cancer development, a great therapeutic
challenge remains. Here, we revisit the birthplace of cancer biology and review
how one of the first discovered oncogenes, RAS, drives cancers in new and
unexpected ways. As our understanding of oncogenic signaling has evolved, it is
clear that RAS signaling is not homogenous, but activates distinct downstream
effectors in different cancer types and grades. RAS signaling is tightly
controlled through a series of post-transcriptional mechanisms, which are
frequently distorted in the context of cancer, and establish key metabolic and
immunologic states that support cancer growth, migration, survival, metastasis,
and plasticity. While targeting RAS has been fiercely pursued for decades, new
strategies have recently emerged with the potential for therapeutic efficacy.
Thus, understanding the complexities of RAS biology may translate into improved
therapies for patients with RAS-driven cancers.




INTRODUCTION

The RAS family represents some of the earliest described oncogenes and its
discovery fundamentally transformed our understanding of cancer biology.
Originally identified in the 1960s as a viral component that induced formation
of sarcomas in rats (1, 2), the RAS oncogenes were later found to be normal
components of the human genome (3, 4) that were capable of transforming normal
human cells (5, 6). Since these early studies, additional work has highlighted
the importance of RAS as a contributor to many human cancers and has more fully
elucidated its signaling axis and molecular regulators. As a small
membrane-localized GTPase, RAS proteins integrate a number of proliferative
signals to establish a tumorigenic cellular circuit when aberrantly activated.
Encoded by the KRAS4A, KRAS4B, HRAS, and NRAS genes, RAS family members are
among the most frequently altered oncogenes in human cancers. In this review, we
dissect the oncogenic circuitry established by RAS and discuss its numerous
roles in supporting proliferative signaling, survival pathways, metabolic and
immunologic functions, and its potential vulnerabilities as a therapeutic
target.


RAS SIGNALING CASCADE AND REGULATION

RAS signaling can be activated by a number of cellular receptors including
receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs), and
integrin family members. These signaling cascades initiate RAS activation
through assembly of several scaffolding proteins that mediate conversion of RAS
from an inactive GDP-bound form to an active GTP-bound state. Epidermal growth
factor receptor (EGFR) is a member of the RTK family and one of the best
characterized activators of RAS signaling through recruitment of the molecular
scaffolding protein growth factor receptor bound protein 2 (GRB2) (7). GRB2
recruits the RAS-guanine exchange factor (RAS-GEF) SOS1, which activates the RAS
protein through a conformational change induced by exchanging GDP for GTP.
Similarly, other RTK family members including platelet derived growth factor
receptor beta (PDGFR-β) can initiate RAS activation through recruitment of GRB2
(8), and colony stimulating factor 1 receptor (CSF-1R) signaling functions
through activation of RAS (9). Several GPCRs also function in a RAS-dependent
manner with the beta-gamma subunit of GPCRs activating RAS signaling (10). GPCRs
activate RAS through stimulation of both non-RTKs (11) (including src, Lyn, and
Syk) and RTKs as described above. Certain downstream signaling functions of
integrin proteins are also RAS dependent (12).

RAS can be further activated by additional RAS-GEFs including the RAS-GRF and
RAS-GRP family members or negatively modulated by a series of RAS-GTPase
activating enzymes (RAS-GAPs), including neurofibromin 1 (NF1) (13). These RAS
activity regulators are also frequently altered across a number of cancer types.
Post-translational modifications are also critical to the functions of the RAS
protein. The addition of an isoprenyl group (farnesylation) by farnesyl
transferase is essential for RAS localization to the plasma membrane and
downstream signaling roles (14). Further, palmitoylation of the NRAS and HRAS
proteins by the enzymes DHHC9 and GCP16 promotes membrane localization and
efficient signaling (15). Continuous cycles of NRAS and HRAS palmitoylation
ensure that these proteins are selectively localized to the Golgi or plasma
membrane and not in other intracellular membranes (16, 17). KRAS, however, can
localize to the plasma membrane without the requirement of palmitoylation (18).
The post-translational membrane anchor that fastens KRAS to the plasma membrane
contains unique sequences and electrostatic properties that determine the
specific localization of RAS nanoclustering within anionic phospholipids (19).
KRAS dimerization is also critical for oncogenic signaling (20).

Further post-translational modifications including mono-ubiquitination favor the
active form of RAS (21, 22), while di-ubiquitination decreases downstream
signaling output through ERK (23). RAS signaling can be abrogated through
ubiquitination by an LZTR1-CUL3 complex, which inhibits its membrane
localization (24, 25). RAS acetylation has also been shown to reduce signaling
activity, with cells dependent on the protein deacetylases HDAC6 and SIRT2 to
maintain RAS signaling (26, 27). Additionally, acylpeptide hydrolase (APEH)
contributes to the appropriate localization of RAS to the plasma membrane by
regulating phosphatidylserines in the plasma membrane (28) (Figure 1).


FIGURE 1

Figure 1. RAS pathway in cancer. This diagram demonstrates (1) the upstream
activators of RAS signaling (2) regulators of RAS membrane localization, (3)
regulators of RAS activity, (4) downstream signaling effector pathways, and (5)
downstream functional effects of RAS signaling in cancers.




Following activation, RAS can execute a variety of functions that promote cancer
development including oncogenic transcription, cell cycle progression, cellular
survival, cell growth and metabolism, and cell motility and migration. First,
RAS activates the mitogen-activated protein kinase (MAPK) pathway defined by a
RAF-MEK-ERK signaling axis. This pathway activates transcription of a number of
proliferative signaling networks driven by FOS, JUN, and ETS family
transcription factors, as well as MYC. These factors support cancer cell
proliferation through promoting cell cycle entry, angiogenesis, and survival.
Second, RAS plays an important role in the activation of the PI3K-AKT signaling
network, which supports oncogenic transcription through NF-κB signaling, evasion
of apoptosis through inhibition of the pro-apoptotic enzyme BAD, and cell growth
and metabolism through mTOR. Third, activation of TIAM1 drives cancer cell
motility and migration through a Rac-Rho and Rac-PAX dependent network. Other
RAS effectors have been studied extensively (29) (Figure 1).

KRAS can also mediate activation of canonical Wnt signaling while suppressing
non-canonical Wnt pathways to promote tumor growth. In APC-deficient colon
cancers, KRAS-dependent cells specifically upregulate BMP signaling, which
activates expression of TAK1/MAP3K7 and downstream transcriptional upregulation
of canonical Wnt target genes. This pathway can be targeted with TAK1 kinase
inhibitors, which selectively ablate KRAS-mutant colon cancer xenografts (30).
KRAS has also been shown to inhibit non-canonical Wnt signaling through
sequestering calmodulin and blocking transcription of the Frizzled 8 receptor, a
G protein-coupled receptor activator of non-canonical Wnt signaling (31). This
represents one distinguishing feature between RAS family proteins, as HRAS is
unable to similarly affect this pathway (31). Because non-canonical Wnt
signaling reduces activation of canonical Wnt signaling pathways, these studies
consistently show that KRAS activates canonical Wnt signaling to support
stem-like properties of cancer cells and tumor growth and that this node may be
targeted for cancer therapy.


ROLE OF RAS MUTATIONS IN DIFFERENT CANCER TYPES

The Cancer Genome Atlas (TCGA) project identified the RTK-RAS signaling pathway
as the most frequently altered oncogenic network in cancer, with 46% of all
samples displaying alterations (32). RAS alterations contribute to 20–30% of all
human cancers. KRAS mutations are exceedingly common in pancreatic
adenocarcinomas and colorectal cancers, while NRAS mutations are more common in
melanomas, thyroid cancers, and leukemias (33, 34) (Figures 2A–C). Although
KRAS, HRAS, and NRAS share functional similarities, KRAS missense
gain-of-function mutations tend to occur on the 12th codon, while those in HRAS
and NRAS occur on the 61st codon and are differentially utilized across cancer
types (33–35) (Figure 2D). These mutations act by creating enhanced RAS
activity, effectively uncoupling pro-proliferative downstream signaling from
growth factor receptors. Alterations in any of these RAS family genes is
associated with poor patient prognosis in pan-cancer analyses (33, 34) (Figure
2E), and RAS pathway gene alterations frequently co-occur with the exception of
KRAS-BRAF and KRAS-NRAS gene pairs, which are mutually exclusive (33, 34)
(Figure 3).


FIGURE 2

Figure 2. Epidemiology of RAS alterations in cancer. (A) Frequency of KRAS
alterations across a number of cancer types. Data were derived from Cerami et
al. (33) and Gao et al. (34). (B) Frequency of NRAS alterations across a number
of cancer types. Data were derived from Cerami et al. (33) and Gao et al. (34).
(C) Frequency of HRAS alterations across a number of cancer types. Data were
derived from Cerami et al. (33) and Gao et al. (34). (D) Localization of RAS
gene mutations across the gene body. Data were derived from Cerami et al. (33)
and Gao et al. (34). (E) Prognosis of cancer patients with or without
alterations in KRAS, NRAS, or HRAS. Data were derived from Cerami et al. (33)
and Gao et al. (34).



FIGURE 3

Figure 3. Gene pair co-occurrence among RAS pathway genes. (A) Co-occurrence
plot RAS pathway genes across a number of cancer types. Data were derived from
Cerami et al. (33) and Gao et al. (34). (B) Gene pair co-occurrence plot of RAS
pathway genes. Blue bars indicate gene pairs that are significantly mutually
exclusive, red bars indicate gene pairs that are significantly co-occurrent, and
black bars indicate gene pairs without significant co-occurrence. (C) Gene pair
co-occurrence network. Solid blue lines indicate gene pairs that are
significantly mutually exclusive, solid red lines indicate gene pairs that are
significantly co-occurrent, and dotted lines indicate gene pairs without
significant co-occurrence.




Pancreatic ductal adenocarcinomas (PDACs) are highly lethal and display
exceptionally high frequency of KRAS mutations (94% mutant). RAS mutations in
PDAC commonly co-occur with CDKN2A mutations and deletions, TP53 mutations, and
SMAD4 mutations (36–38). Colorectal cancers are largely initiated by mutations
in APC, which lead to uncontrolled Wnt signaling, followed by loss of function
of TP53, inactivation of TGF-β signaling, and mutations in KRAS in ~37% of cases
(39). KRAS is the most commonly mutated oncogene in lung adenocarcinoma,
occurring in 33% of cases, along with EGFR, BRAF, and TP53 mutations (40).
Despite the high prevalence of KRAS mutations and RTK activation in lung
adenocarcinomas (and other forms of non-small cell lung cancers), small cell
lung carcinomas are characterized by nearly universal inactivation of TP53 and
RB1 through mutation or deletion, without alterations in RAS (41). In contrast
to pancreatic, lung, and colon cancers, melanomas contain NRAS mutations in
20–30% of cases (42) NRAS is also commonly mutated in acute myeloid leukemias in
15% of cases (43, 44).

The differential mutation rate across cancers suggests that each mutational
event may activate distinct signaling events and that each tissue type may be
differentially poised to transform following RAS mutation. For example, HRAS
displayed a greater capacity to transform fibroblasts than the other RAS family
members (45), while in hematopoietic cell models, NRAS demonstrated a stronger
transforming potential (46). RAS family members display distinct
post-translational modifications, which regulate their subcellular localization
and differential signaling preferences, which have been extensively reviewed
elsewhere (47–49).


RAS AND METABOLISM

Dysregulated metabolism is a key hallmark of cancer, and activation of RAS
signaling supports cancer initiation, maintenance, and progression through
driving altered metabolic networks. RAS signaling promotes oncogenic metabolism
by coordinating numerous metabolic processes including lipid, nucleotide, and
glycolytic pathways. Specifically, RAS signaling supports cellular bioenergetic
needs and enhances glucose uptake through induction of the GLUT1 glucose
transporter promoting survival in low-nutrient conditions and increased
glycolytic metabolism (50). This glucose is shunted away from the tricarboxylic
acid (TCA) cycle to support glycolytic metabolism, protein glycosylation, and
nucleotide metabolism through the pentose phosphate pathway (51, 52). Cells also
upregulate glutamine metabolism and the phosphoserine biosynthetic pathway
through upregulation of biosynthetic enzymes in these pathways (53). KRAS
redirects glutamine utilization to support cellular redox balance through
transcriptional regulation of the GOT1 (glutamic-oxaloacetic transaminase 1)
enzyme and creates a dependency on glutamine metabolism (54). Co-mutation of
KRAS with loss of KEAP1 (kelch like ECH associated protein 1) further extended
the glycolytic phenotype, dependence on glutamine, and sensitivity to
glutaminase inhibitors in lung adenocarcinoma models (55). RAS signaling also
acts to support nucleotide biosynthesis via MYC activation. RAS upregulates MAPK
signaling, which induces MYC and drives nucleotide metabolism through the
pentose phosphate pathway (56).

Increased copy number of mutant oncogenic KRAS that typically occurs later in
the process of tumorigenesis further activates glycolytic metabolism and
supports glutathione synthesis, but can also direct metabolites into the TCA
cycle in lung cancer cells to support tumor progression (57). This mitochondrial
metabolism has been shown to be essential for anchorage-independent cell growth
in KRAS-driven cancers by promoting generation of reactive oxygen species, which
modulate ERK signaling (58). This suggests that differential dosage of KRAS
expression can have contrasting effects on cellular metabolism and highlights
the evolution of metabolic states throughout tumor development. RAS allelic
imbalance and loss of wild-type KRAS alleles can further extend the oncogenic
properties of cancer cells and mark the most aggressive undifferentiated cells
(59), but also create a dependency on the MAPK signaling pathway with unique
sensitivities to pharmacologic MEK inhibition (60).

While cancers rely heavily on endogenous synthesis of substrates for anabolic
needs, RAS-driven cancers also utilize mechanisms to recover materials from
their extracellular environments in the form of micropinocytosis (61, 62). This
process supports cancer cell growth through scavenging extracellular amino acids
for use in protein synthesis, and glutamine for a variety of metabolic processes
(63). RAS activation can also support cell membrane biosynthesis through fatty
acid uptake from lysophospholipids in the surrounding microenvironment, reducing
dependence on endogenous lipid synthesis (64). KRAS signaling sustains cancer
cells under conditions of nutrient stress by activating an NRF2-ATF4 axis to
increase amino acid transport and protein biosynthesis, preventing apoptotic
cell death through increased asparagine synthase activity (65).

Despite this metabolic resiliency through increased nutrient scavenging
capacity, RAS driven cancers are dependent on autophagy, which is essential for
mitochondrial recycling and oxidative capacity (66). Autophagy is essential for
proper mitochondrial function and nucleotide synthesis in KRAS-driven tumors
(67), as well as for efficient catabolism of fatty acids (68). In RAS driven
pancreatic cancers, autophagy is supported by the MiT/TFE family of
transcription factors, including MITF, TFE3, and TFEB, which activate genes that
promote autophagy and lysosomal pathways to maintain intracellular amino acid
pools (69). The acyl-CoA synthetase family member, ACSL3, whose expression is
driven by mTOR signaling downstream of RAS, specifically regulates intracellular
fatty acid metabolism and utilization in RAS-dependent cancers by supporting
fatty acid uptake, accumulation, and β-oxidation (70). Interestingly, RAS-driven
metabolic dependencies can also be tissue- and context-dependent. Branched-chain
amino acid metabolism is a key dependency in KRAS-driven non-small-cell lung
carcinoma (NSCLC) cells in which they are essential for non-essential amino acid
and DNA synthesis. However, these metabolic circuits are dispensable in
KRAS-driven pancreatic ductal adenocarcinoma (PDAC) cells (71) (Figure 4).


FIGURE 4

Figure 4. The RAS pathway orchestrates cellular metabolism. This diagram depicts
metabolic pathways that are altered in RAS-driven cancers.





RAS IN CANCER METASTASIS

In addition to driving processes essential for early phases of tumorigenesis,
RAS activity is important for the acquisition of more malignant features,
including supporting metastasis. In mouse models of colorectal cancer, while
primary tumors were characterized by a heterogeneous population of cells bearing
both oncogenic KRAS mutations and wild-type KRAS, metastatic sites were largely
comprised of more uniform cell populations harboring oncogenic KRAS (72). This
metastatic phenotype was promoted by transforming growth factor beta (TGF-β)
signaling (72). Distinct from heterogeneity in cellular populations with respect
to KRAS mutation status, acquisition of multiple oncogenic KRAS mutations within
single cells through focal amplifications and loss of the wild-type allele (loss
of heterozygousity) can promote tumor metastasis and aggressive properties (59).
KRAS also supports metastatic dissemination through repression of Raf Kinase
Inhibitory Protein (RKIP), a putative tumor suppressor with roles in cell
migration, motility, and epithelial-to-mesenchymal transition (73). Activation
of KRAS signaling along with homozygous deletion of LKB1 (also known as STK11 or
serine/threonine kinase 11) promoted cancer progression and metastasis in
non-small cell lung cancer models (74). In KRAS-driven pancreatic cancer models,
deletion of LKB1 enhanced the tumorigenicity and proliferation rate of cancer
cells through enhanced serine biosynthesis and S-adenosyl-methionine (SAM),
which supports DNA methylation (75).


RAS AND THE IMMUNE SYSTEM

Interactions between cancer cells and the immune system are essential features
of cancer biology. In order to survive and thrive, cancer cells must avoid
immunoediting by immune effector cells; however, cancer cells also frequently
gain proliferative advantage from the surrounding immune microenvironment (76).
RAS signaling reduces expression of MHC class I molecules on the surface of
cancer cells, rendering them less vulnerable to immune-mediated cell death by
cytotoxic T-cells (77, 78). Immune checkpoints such as PD-L1 (CD274) serve to
dampen the reactivity of the immune system and to prevent autoimmunity. Cancers
frequently subvert this mechanism to avoid being targeted by the immune system.
RAS signaling can promote this effect in an MEK-dependent manner by stabilizing
PD-L1 mRNA through downregulation of tristetraprolin (TTP/ZFP36), an RNA binding
protein which typically degrades mRNAs (79). These findings may partially
explain the observation that KRAS mutant non-small cell lung cancer patients
display better responses to PD-1 inhibition with nivolumab than KRAS wild-type
patients (80, 81). In hepatocellular carcinoma models, dual KRAS and MYC
signaling can translationally enhance PD-L1 levels by bypassing upstream open
reading frames, which typically serve a repressive role (82). This contributes
to a more aggressive and metastatic phenotype with the capacity to evade the
immune system. KRAS and MYC signaling further cooperate to promote the
development of aggressive and invasive adenocarcinomas by recruiting
immunosuppressive macrophages via the chemokine CCL9 and excluding T-cells and
NK cells via interleukin-23 (IL-23) (83). These alterations allow developing
tumors to evade immune-mediated attack. In lung cancer models, KRAS supports
expression of IL6-mediated chronic inflammation, which reorganizes the tumor
microenvironment by recruiting myeloid derived suppressor cells (84, 85).
Targeting MAPK and CDK4/6 pathways in RAS mutant lung cancer cells leads to
natural-killer (NK) cell-mediated attack of tumor cells through induction of
senescence pathways (86). Activation of the MEK/ERK signaling pathway by the
oncogenic KRAS G12D mutation increases secretion of IL-10 and TGF-β from
pancreatic cancer cells, which promotes conversion of T-cells to an
immunosuppressive regulatory T-cell (Treg) state (87). Additionally, co-mutation
with STK11 is associated with a reduction in NF-κB signaling in RAS mutant
tumors and suppression of tumor immunosurveillance while co-mutation with TP53
is associated with increased immune responses (88). This suggests that mutations
commonly co-occurring with RAS impinge upon the immune reactivity of RAS driven
cancers.

Besides avoiding immune-mediated destruction, cancer cells frequently benefit
from a proinflammatory microenvironment that sustain oncogenic processes. In
pancreatic intraepithelial neoplasia models of pancreatic cancer precursor
lesions, KRAS signaling induced expression of IL-17 receptors on preneoplastic
cells and infiltration by IL-17 secreting T-cells, both of which accelerated
progression to a neoplastic state (89). RAS signaling also promotes tumor
vascularization and inflammation by inducing secretion of IL-8 from cancer cells
through MAPK and PI3K pathways (90). Tumor vascularization is further driven by
KRAS-mediated induction of hypoxic HIF signaling, which drives expression of
vascular endothelial growth factor (VEGF) (91). KRAS activation can activate
inflammatory processes in lung cancer models by stimulating accumulation of
macrophages and neutrophils through production of inflammatory chemokines (92)
(Figure 5).


FIGURE 5

Figure 5. The RAS pathway shapes interactions between cancer cells and the
immune microenvironment. This diagram depicts mechanisms by which RAS signaling
promotes cancer through (1) supporting cancer cell immune evasion and (2)
driving immune-mediated stimulation of cancer cell growth.





THERAPEUTIC TARGETING OF RAS

Because of the numerous ways in which RAS activity supports tumor cell
proliferation, survival, metabolism, microenvironmental interactions, and immune
evasion, efficient therapeutic targeting of RAS has been the focus of a large
body of research. While it was previously believed that RAS is an undruggable
target due to its molecular structure, new insights into its biological
functions and molecular regulators may allow for efficient pharmacological
inhibition of RAS effectors and discoveries of synthetic lethality.


DIRECT RAS INHIBITORS

Direct inhibition of oncogenic RAS could be a powerful therapeutic approach to
ablate RAS-driven tumors. Studies of the molecular structure of the common KRAS
G12C variant have informed the development of specific inhibitors that
selectively target the mutant form of KRAS and both limit its activation by
favoring binding to GDP as well as blocking its downstream signaling through RAF
(93). Another compound targeting the KRAS G12C variant, ARS-853, selectively
reduced the frequency of the active, GTP-bound KRAS, and inhibited cell
proliferation in lung cancer models and suggests that nucleotide cycling between
GDP and GTP bound forms are essential for its molecular functions (94, 95).
Next-generation forms of KRAS G12C targeting agents, including ARS-1620,
demonstrated improved potency compared to earlier generation agents and block
oncogenic RAS signaling and tumor growth in vivo in a target-specific manner in
non-small cell lung cancer models (96). These agents have been extensively
reviewed elsewhere (97, 98). In addition to mutation-specific RAS inhibitors,
pan-RAS inhibitors that target HRAS and NRAS as well as KRAS have been
developed. One of these pan-RAS inhibitors, compound 3,144, efficiently silenced
PI3K-AKT and MEK-ERK signaling downstream of RAS and prevented growth of
RAS-driven xenograft cancer models. However, some off-target effects and
toxicities apparent in this first-generation compound have prevented wide-spread
clinical adoption at this time (99). To advance rational design of compounds
with RAS targeting potential, computational modeling of RAS three-dimensional
structure revealed conformational changes that occur during RAS deactivation,
suggesting that stabilizing these inactive forms may reduce RAS signaling
efficacy (100). Similar efforts identified a high-affinity allosteric KRAS
inhibitor that impairs KRAS signaling and cancer cell growth in cells bearing
several distinct types of KRAS activating mutation (101). Detailed
conformational dynamics analyses and structural biology approaches uncovered
numerous vulnerabilities and co-dependencies of the RAS enzyme, which may be
exploited for therapeutic targeting and which have been detailed extensively
elsewhere (102, 103).

In addition to small molecule inhibitors, other therapeutic approaches have
investigated methods to deliver nucleic acid-based delivery of therapeutic
compounds to cancer cells in vivo. Using nanoliposomal delivery of
KRAS-targeting siRNAs, KRAS mRNA expression could be dramatically reduced with
subsequent decrease in tumor growth and metastatic potential in colon and lung
cancer models (104). Nanoliposomes can also be used to deliver miRNAs that
specifically target KRAS and impair tumor growth and metastasis in lung cancer
models (105). Cyclodextrin polymer nanoparticles can also be used to deliver
siRNAs to cancer cells in vivo. Optimized siRNAs targeting KRAS impaired colon
cancer growth in vivo while combinatorial inhibition of KRAS and PIK3CA/PIK3CB
significantly improved tumor control compared to single agents alone,
demonstrating that targets can be effectively multiplexed (106). In contrast to
liposomal or other nanoparticle technologies, exosome-mediated delivery of
siRNAs have greater efficiency due to longer persistence in the circulation and
take advantage of RAS-mediated upregulation of micropinocytosis for greater
uptake by RAS-driven cells. Exosomal delivery of siRNAs targeting KRAS reduced
expression of KRAS, suppressed tumor formation, and inhibited metastatic
progression in mouse pancreatic cancer models (107).


INHIBITORS OF RAS MODULATORS

Besides directly targeting the enzymatic domain of RAS, many studies have
investigated targeting its subcellular localization. As described previously,
RAS relies on a number of factors for post-translational modifications and
localization to the cell membrane. The phosphodiesterase PDE-delta binds to
farnesylated RAS and promotes its efficient signaling by selectively localizing
RAS to the plasma membrane as opposed to intracellular membranes (108).
Inhibition of the interaction between PDE-delta and KRAS disrupted RAS
localization and signaling and impaired cell proliferation in pancreatic cancer
models (109). Additionally, inhibition of the lysophospholipase APT1 with
palmostatin B blocked RAS depalmitoylation and impaired RAS localization and
signaling efficacy and contributed to re-acquisition of contact inhibition in
HRAS-transformed fibroblasts (110). This inhibitor demonstrated similar effects
in NRAS-driven hematologic cancer models (111). Farnesyltransferases are also
essential for RAS membrane localization and represent therapeutic targets.
Several of these agents have shown promise in clinical trials by disrupting RAS
signaling in combination with other therapeutic agents (112–114), although these
effects may be based on inhibition of other farnesylation-dependent enzymes
beyond RAS. RAS geranylgeranylation following inhibition of farnesyltransferases
reactivates RAS signaling and serves as a common resistance mechanism (115).
Combinatorial targeting of farnesyl and geranylgeranyltransferases may overcome
this resistance (116).

SOS is a RAS-specific guanine exchange factor (GEF) that mediates the conversion
of RAS from an inactive GDP-bound state to an active GTP-bound state. Because of
this important role in regulating RAS activity, SOS is a natural target for RAS
driven cancers. Helical proteins that interrupt the RAS-SOS interaction blocked
RAS activation and downstream ERK activity following EGFR stimulation (117).
Additional studies have identified small molecules that can interrupt the
RAS-SOS interaction and disrupt RAS activation and downstream MAPK and PI3K
signaling (118, 119). In order to mediate its downstream effects, RAS binds to a
series of effector molecules through a RAS binding domain. Inhibition of this
RAS binding domain with the small molecule agent rigosertib impairs the
interaction between RAS and RAF, as well as Ral and PI3K, simultaneously
incapacitating several downstream RAS effectors and impairing tumor growth in
vitro and in vivo (120). RAS also relies on kinase suppressor of ras (KSR),
which serves as a scaffolding factor that links RAS to RAF and allows for MEK
activation (121–124). Stabilization of the inactive form of KSR with small
molecule compounds blocked this signal transduction from RAS to RAF and enhanced
efficacy of MEK inhibitors (125). STK19 activates oncogenic signaling in
melanoma cells through selective phosphorylation of mutant NRAS, which supports
its interaction with downstream effectors through the RAS binding domain.
Pharmacologic inhibitors of STK19 blocked NRAS phosphorylation and impaired
melanoma cell growth and tumor formation capacity, and extended survival of
tumor-bearing mice (126).

RAS can also be activated by the protein tyrosine phosphatase SHP2 (encoded by
the PTPN11 gene). SHP2 binds to receptor tyrosine kinase growth factor receptors
through its SH2 domain and mediates activation of RAS through dephosphorylation
of RAS, increasing its association with RAF (127, 128). Inhibition of the SHP2
phosphatase domain with a small molecule inhibitor suppressed RAS signaling and
impaired proliferation of receptor tyrosine kinase-driven cancer cells in vitro
and in vivo, although RAS-mutant cells were not sensitive to this drug in vitro
(129). Targeting SHP2 further sensitized pancreatic cancer cells to MEK
inhibition and promoted a senescence response in KRAS-mutant non-small cell lung
cancer models under nutrient-restricted conditions (130, 131). These findings
suggest that combinatorial targeting of signaling elements upstream and
downstream of RAS may be a useful therapeutic approach.


INHIBITION OF DOWNSTREAM SIGNALING AND RESISTANCE MECHANISMS

Aberrant RAS activation can also be targeted through inhibition of downstream
signaling elements, such as MEK. Despite these efforts, targeted therapies are
frequently plagued by the robust emergence of resistance. In KRAS mutant
cancers, targeting of MEK with trametinib led to compensatory signaling through
fibroblast growth factor receptor 1 (FGFR1). Combinatorial therapy using
trametinib and FGFR1 inhibition effectively abolished this resistance mechanism
and served as a useful combinatorial strategy (132). RAS-driven cancer cells
could further overcome MEK inhibition through overexpression of ERBB3. Targeting
the related RTKs EGFR and ERBB2 reversed this effect and sensitized to MEK
inhibitors (133). Targeting RAF kinases can also reverse resistance to MEK
inhibitors through downregulation of MAPK signaling (134). MEK inhibitors
further drive compensatory activating phosphorylation of the KSR-1 scaffolding
protein, which promotes PI3K-AKT signaling that circumvents inhibition of RAS
signaling effectors (135). In the context of RAF or MEK inhibition, YAP1, a
component of the Hippo pathway, promoted survival of RAS-mutant cells, with
combinatorial inhibition of MEK and YAP1 yielding improved therapeutic efficacy
(136). Thus, development of therapeutic resistance following RAS inhibition is
exceedingly common. Greater understanding of these resistance mechanisms may
allow researchers to collapse the great degree of cellular plasticity in these
signaling networks through combinatorial inhibition of survival and escape
pathways.

Despite our detailed understanding of the major RAS downstream signaling
elements in cancers, recent evidence revealed that the temporal dynamics of
signal transduction, and not just the pathway constituents themselves, are
critical to the resulting biological effects. Because of this phenomenon,
treatment with BRAF inhibitors may have counterproductive effects on RAS
signaling by prolonging the typically short pulses of RAS activity into long
periods of downstream ERK activation (137). Furthermore, while BRAF inhibitors
are effective in blocking growth of cancer cells driven by the BRAF-V600E
mutation, BRAF inhibition paradoxically activates MAPK signaling in KRAS mutant
tumors through inducing increased dimerization of BRAF with RAS (138). Because
the complexity of these signaling pathways has not been completely elucidated,
caution must be used when developing therapeutic agents and their downstream
effects must be empirically determined.


IDENTIFICATION OF RAS-SPECIFIC SYNTHETIC LETHALITY

In addition to targeting RAS signaling directly through its enzymatic activity
or indirectly through its regulators or downstream signaling effectors,
therapeutic targeting of dependencies established by oncogenic RAS is a
promising approach. The unique cellular states established by RAS activation
create new nodes of fragility that may be amenable to anti-cancer therapies.
Increased RAS copy number engages a glycolytic switch which increases glycolysis
and shifts glucose utilization toward the TCA cycle and glutathione synthesis.
These metabolic changes create sensitivity to glutathione synthesis inhibitors
(57). Loss of wild-type RAS further sensitized cells to MEK inhibition,
suggesting that allelic imbalance at the KRAS locus can impact dependency on
downstream signaling elements (60). Additionally, increased levels of the GLUT1
glucose transporter facilitates selective sensitivity of RAS driven cancers to
vitamin C, the oxidized version of which is preferentially imported, depleting
intracellular glutathione, and generating oxidative stress (139). Other
targeting approaches have leveraged oxidative stress to selectively ablate NF1-
or KRAS- mutant tumors through combinatorial therapy with HDAC and mTOR
inhibitors, which suppress glutathione synthesis and the thioredoxin antioxidant
pathway (140). These findings suggest that RAS driven cancers are particularly
vulnerable to oxidative damage and are unable to efficiently cope with oxidative
stress. KRAS-driven cancers employ micropinocytosis to scavenge nutrients from
the extracellular environment. Through interacting with cell surface integrins,
the carbohydrate binding protein galectin-3 mediates formation of macropinosomes
and reduces reactive oxygen species by recruiting KRAS clusters on the cell
membrane to promote RAS signaling. This event can be effectively targeted with
galectin-3 inhibitors (141).

Whole genome shRNA and CRISPR screening strategies have identified RAS-specific
synthetic lethalities, elucidating potential novel therapeutic targets. Cells
with oncogenic RAS rely on TBK1, an IκB kinase, to activate NF-κB signaling to
prevent apoptosis (142). KRAS-driven non-small cell lung cancers also rely on
the nuclear export receptor XPO1, which clears nuclear IκBα and supports NF-κB
activity. KRAS-mutant models are selectively sensitive to small molecule
inhibition of XPO1 (143). RAS mutant non-small cell lung cancers are
specifically dependent on GATA2, a transcription factor that regulates the
proteasome, Rho signaling pathways, and maintenance of NF-κB signaling via the
IL-1 pathway (144). Collectively, these results point toward NF-κB signaling as
an essential pro-survival signal selectively utilized by RAS driven cancers.
Furthermore, the protein kinase STK33 is a RAS-dependent essential factor that
inhibits mitochondrial apoptosis downstream of S6-kinase (S6K1) signaling (145).
In the context of MEK inhibition, the mitochondrial anti-apoptotic gene BCL-XL
is essential in RAS-driven cancers. Combinatorial inhibition of BCL-XL with MEK
signaling enhanced cell death in colorectal, lung, and pancreatic cancers
bearing RAS mutations, suggesting that BCL-XL displays a synthetic lethal
interaction with RAS in a context-specific manner (146).

Other screens have demonstrated increased dependence on ribosomal biogenesis and
translational control, protein neddylation, protein sumoylation, RNA splicing
pathways, and mitotic control in RAS mutant cancer models (147). PLK1, a kinase
involved in centrosome maturation and spindle assembly during mitotic
progression, was specifically essential and targeting this kinase with a small
molecule inhibitor selectively targeted RAS mutant cells. This dependence on
mitotic machinery and sensitivity to mitotic stress was specific to RAS-mutant
cells when compared to PIK3CA driven cells (147). The cell cycle regulator CDK4
also displays a synthetic lethal relationship with KRAS in non-small cell lung
cancers (148). A guanine nucleotide exchange factor for Rac family GTPases,
PREX1, is essential for MAPK activation in RAS mutant acute myeloid leukemias,
and cells driven by oncogenic RAS were sensitized to Rac/PAK family inhibitors
(149).


IMMUNOTHERAPIES

Therapeutic approaches that harness the immune system to target cancers have
emerged as an effective strategy. Recently, CD8+ T-cells have been isolated from
a patient with metastatic colorectal cancer that specifically recognize mutant
KRAS. Ex vivo expansion of this population followed by reinfusion into the
patient led to reduction in metastatic burden, suggesting that immunotherapeutic
approaches may be effective in targeting RAS (150, 151). Further
immunotherapeutic efforts have utilized T-cell receptors engineered to
specifically target oncogenic forms of KRAS to control tumor growth in
pancreatic cancer models (152) In addition to direct targeting of RAS antigens,
immunotherapeutic approaches have been explored in combination with inhibition
of downstream RAS signaling elements. In BRAF-driven melanomas, combination of
BRAF, MEK, and immune checkpoint inhibition through PD-L1 inhibitors enhanced
cancer cell death and displayed efficacy in early clinical trials for metastatic
melanoma (153, 154). Combinations of MEK and BRAF inhibitors with PD-L1
inhibitors demonstrated some promise in metastatic colorectal cancers and
melanomas in early clinical trials (155, 156). PI3K signaling downstream of RAS
controls interactions between cancer cells and the immune microenvironment.
While overactive PI3K signaling driven by PTEN mutations reduced T-cell-mediated
cytotoxicity, treatment with a PI3Kβ inhibitor enhanced the efficacy of anti-PD1
antibodies in melanoma models (157) (Figure 6).


FIGURE 6

Figure 6. Therapeutic targeting of RAS in cancer. This diagram depicts several
strategies to therapeutically target RAS driven cancers.





CONCLUSIONS

RAS family members are some of the most commonly altered genes in cancer.
Perturbations of RAS signaling establish robust oncogenic circuits that drive
tumor initiation, progression, growth, and survival. Despite our deep knowledge
of the direct downstream signaling effectors of the RAS pathway, continued
exploration has revealed new insights into the similarities and differences
between RAS family members and their preference for particular cancer types.
These efforts have also uncovered the more distal downstream consequences of RAS
signaling across cancers, including its rewiring of cellular metabolism and
capacity to unlock nutrient scavenging pathways, its role in metastasis, and its
dual role in regulating the immune microenvironment. These processes endow
cancer cells with the plasticity required for survival in dynamic conditions,
but also create key vulnerabilities, which can be therapeutically targeted
through a number of avenues. Taken together, a deeper understanding of RAS
biology will critically inform clinical care and serves as a model for
interrogation of other driver alterations in cancer.


AUTHOR CONTRIBUTIONS

RG and XW contributed to the conception, research, writing, editing, and design
of figures for this manuscript.


CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential
conflict of interest.


ACKNOWLEDGMENTS

This work was supported by grants provided by NIH: CA217065 (RG). Figures were
prepared in part using images from Servier Medical Art by Servier
(https://smart.servier.com/), which is licensed under a Creative Commons
Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).


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Keywords: RAS, cancer, metabolism, immunology, mitogen activated kinase, cancer
therapy

Citation: Gimple RC and Wang X (2019) RAS: Striking at the Core of the Oncogenic
Circuitry. Front. Oncol. 9:965. doi: 10.3389/fonc.2019.00965

Received: 17 August 2019; Accepted: 11 September 2019;
Published: 24 September 2019.

Edited by:

Georgia Konstantinidou, University of Bern, Switzerland

Reviewed by:

Roberto Giovannoni, University of Pisa, Italy
Lu Shaoyong, Shanghai Jiao Tong University, China

Copyright © 2019 Gimple and Wang. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not
comply with these terms.

*Correspondence: Xiuxing Wang, xiuxingwang81@163.com



Disclaimer: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or
those of the publisher, the editors and the reviewers. Any product that may be
evaluated in this article or claim that may be made by its manufacturer is not
guaranteed or endorsed by the publisher.



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