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 Conquering RAS
From Biology to Cancer Therapy

Edited by

Asfar S. Azmi
Department of Oncology, Wayne State University School of Medicine,
Karmanos Cancer Institute, Detroit, MI, USA

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Dedication

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survivor, inspirational personality and hope for those fighting
the disease.
List of Contributors

A.S. Azmi Wayne State University, Detroit, MI, United States

W. Bai University of South Florida, Tampa, FL, United States
G. Bepler Karmanos Cancer Institute, Detroit, MI, United States
M. Bian Eastern Virginia Medical School, Norfolk, VA, United States
J.K. Bruflat Cellular and Molecular Immunology Laboratory, Rochester, MN,
United States
H.-H. Chang David Geffen School of Medicine at UCLA, Los Angeles, CA,
United States
C.R. Chow Northwestern University, Chicago, IL, United States
G.J. Clark University of Louisville, Louisville, KY, United States
K. Ebine Northwestern University, Chicago, IL, United States
G. Eibl David Geffen School of Medicine at UCLA, Los Angeles, CA, United
States
J.L. Eisner Eastern Virginia Medical School, Norfolk, VA, United States
N.S. Gray Dana Farber Cancer Institute, Boston, MA, United States
H.Z. Hattaway Northwestern University, Chicago, IL, United States
J.C. Hunter The University of Texas Southwestern Medical Center at Dallas,
Dallas, TX, United States
J.A.E. Irving Newcastle University, Newcastle upon Tyne, United Kingdom
A.J. Isbell Eastern Virginia Medical School, Norfolk, VA, United States
D.F. Kashatus The University of Virginia School of Medicine, Charlottesville,
VA, United States
A.B. Keeton University of South Alabama Mitchell Cancer Institute, Mobile,
AL, United States; ADT Pharmaceuticals, Inc., Orange Beach, AL, United States
K. Kumar Northwestern University, Chicago, IL, United States; Jesse Brown
VA Medical Center, Chicago, IL, United States
G.A. McArthur Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia;
University of Melbourne, Parkville, VIC, Australia
J.N. Mezzanotte University of Louisville, Louisville, KY, United States
H.G. Munshi Northwestern University, Chicago, IL, United States; Jesse
Brown VA Medical Center, Chicago, IL, United States
M.M. Njogu Eastern Virginia Medical School, Norfolk, VA, United States
E. O’Neill University of Oxford, Oxford, United Kingdom
J.J. Odanga Eastern Virginia Medical School, Norfolk, VA, United States
P.A. Philip Wayne State University, Detroit, MI, United States xiii
xiv List of Contributors

G.A. Piazza University of South Alabama Mitchell Cancer Institute, Mobile,

AL, United States; ADT Pharmaceuticals, Inc., Orange Beach, AL, United States
A.D. Rao Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia;
University of Melbourne, Parkville, VIC, Australia
A.A. Samatar TheraMet Biosciences LLC, Princeton Junction, NJ, United
States
A. Schmidt David Geffen School of Medicine at UCLA, Los Angeles, CA,
United States; Universitätsklinikum Freiburg, Freiburg, Germany
R.L. Schmidt Upper Iowa University, Fayette, IA, United States
L.L. Siewertsz van Reesema Eastern Virginia Medical School, Norfolk, VA,
United States
D.D. Stuart Novartis Institutes for Biomedical Research, Cambridge, MA,
United States
E. Svyatova Eastern Virginia Medical School, Norfolk, VA, United States
A.H. Tang Eastern Virginia Medical School, Norfolk, VA, United States
A.M. Tang-Tan Princess Anne High School, Virginia Beach, VA, United States
R.E. Van Sciver Eastern Virginia Medical School, Norfolk, VA, United States
K.D. Westover The University of Texas Southwestern Medical Center at
Dallas, Dallas, TX, United States
S. Xiang University of South Florida, Tampa, FL, United States
X. Zhang Karmanos Cancer Institute, Detroit, MI, United States
V. Zheleva Eastern Virginia Medical School, Norfolk, VA, United States
Acknowledgments

I would like to especially thank the editorial team at Elsevier for their help
in the publication process. Special thanks to the peer reviewers and technical
staff who helped improve the chapters. The efforts of Lisa Eppich are gratefully
acknowledged. Her help was invaluable during the entire production process.
Last but not least, all of the co-authors are acknowledged for their important
contributions to this book.

xv
Introduction

Ras gene mutations are observed in more than 30% of all cancers and are more
prevalent in some of the difficult-to-treat malignancies, such as >90% in pan-
creatic cancer and also in lung and colon cancers. Ras proteins (N-Ras, H-Ras,
and K-Ras) act as molecular switches that when activated, through binding of
GTP, initiate a cascade of signaling events controlling important cellular pro-
cesses such as proliferation and cell division. A precise and recurring cycling
of GTP to GDP (inactive state) occurs through the intrinsic GTPase activity
of ras. However, mutations in ras result in the loss of this intrinsic GTPase
activity rendering the protein in a constantly activated state. In this scenario, a
continuous signaling from ras results in cells growing uncontrollably, evading
cell death mechanisms and also becoming resistant to therapies. These facts
are well known for the past 30 years; nevertheless, till date strategies to block
ras mutation-driven signaling remain futile. The reasons for such failures have
been attributed to many factors. Chief among them is the lack of any possi-
ble druggable pocket within the ras structure for optimal attachment of small
molecule drugs. In addition, the inherent affinity of ras to GTP (in the picomo-
lar range) restricts the design of high-affinity drugs to displace GTP. Research-
ers have evaluated the benefits of targeting important upstream (EGFR and
IGFR) and downstream (RAF, MEK, and AKT) and other signaling molecules.
With few exceptions, sadly, none of these targets have proven to be effectual
in the clinical setting. The redundancies and cross talk within the associated
pathways pose additional hurdles making the ras fortress impenetrable. Col-
lectively, these multitude number of challenges have led to a sort of consensus
that the ras protein itself in un-druggable.
Thanks to the National Cancer Institute (NCI) ras Initiative, there is a renewed
spark in the field of ras research. Ras Initiative is a concerted and broad-spectrum
approach to ras biology with a single goal and that is to develop effective ther-
apies against this important master cancer regulator. When such an initiative
is underway, a book specifically focused on ras biology becomes an important
resource for researchers working in the field. This is especially important given
that the literature on ras is distributed in the web of knowledge sometimes out xvii
xviii Introduction

of the reach of the most avid researchers working in the field. With this goal in
mind, I have designed this book to bring forward some of the newer topics in
the field of ras biology under one volume. The book is divided into two parts.
Part I deals with ras biology and in Part II many novel therapeutic approaches
are highlighted. Each chapter carries a comprehensive list of up-to-date refer-
ences that are surely going to find their way in the libraries of ras researchers.
Unlike before, in this book, an attempt has been made to accommodate some
of the most burning topics such as ras metabolic vulnerabilities, impact on
microenvironment, role in stemness, effect of post-translational mechanisms,
and the biology of effectors. On the therapeutic side, some very new targets
and novel agents have been presented that will surely make for an interesting
reading.
It is recognized that aside from the contributors of this book there are many
additional groups working in the field. Therefore, every effort has been made
to include the vast library of important references extracted from major contri-
butions across a wide spectrum of related research papers. It was my pleasure
collecting these novel ideas from so many experts working in the field who
have a single goal and that is to conquer ras.

Asfar S. Azmi, PhD

Department of Oncology
Wayne State University School of Medicine
Karmanos Cancer Institute
Detroit, MI 48201, USA
 C H AP TER 1

Ras and RASSF Effector Proteins

J.N. Mezzanotte, G.J. Clark
University of Louisville, Louisville, KY, United States

INTRODUCTION CONTENTS
Ras is the most frequently activated oncoprotein in human cancer. When we
Introduction����������� 3
consider the prevalence of activating point mutations in Ras in tumors combined The RASSF Family of
with the frequent inactivation of GTPase-activating proteins, it seems likely that Proteins�������������������������3
the majority of human cancers use Ras activation as a driving force [1]. In con-
Ras and RASSF1��� 4
trast, the RASSF1A tumor suppressor appears to be the most frequently inacti-
vated tumor suppressor in human cancer [2]. Not only is RASSF1A subjected to Ras and RASSF5
frequent epigenetic inactivation in human tumors but also it can be inactivated (NORE1)����������������� 8
by protein degradation or point mutation at a significant frequency. RASSF1A Ras and RASSF2� 10
contains a Ras-association (RA) domain and can bind directly to Ras [2]. Thus Ras and RASSF3� 11
the most frequently activated oncoprotein in human cancer forms a complex
with the most frequently inactivated tumor suppressor. Ras and RASSF4
(AD037)���������������� 12
Although the mechanistic basis of the potent transforming effects of activated Ras and RASSF6� 12
Ras is well documented, it has also become apparent that Ras activation can
stimulate signaling pathways that suppress growth and survival [3]. For exam- Effects of RASSF
Proteins on
ple, in primary cells, introduction of an activated Ras gene tends to promote
Mitogenic Ras
apoptosis or senescence, not transformation [3–5]. The signaling pathways Effectors�������������� 13
involved in these anti-transformation events are only now being understood.
Therapeutic
Many of them appear to involve the RASSF family of Ras effector/tumor sup-
Ramifications������ 13
pressors. Thus RASSF proteins may serve as Ras death effectors, and their inac-
tivation may enable Ras-dependent tumors to progress to malignancy. Conclusion���������� 15
Glossary�������������� 16
The RASSF Family of Proteins
The RASSF family of proteins consists of 10 members, all of which contain a
Ras-association, or RA, domain, hence the term RASSF: Ras-association domain
family. RASSF1 through RASSF6 have their RA domain toward the C-terminus,
whereas RASSF7 through RASSF10 all have an N-terminal RA domain. The
C-terminal RASSF proteins have been more widely studied and have shown 3
Conquering RAS. http://dx.doi.org/10.1016/B978-0-12-803505-4.00001-1
Copyright © 2017 Elsevier Inc. All rights reserved.
4 CHAPTER 1: Ras and RASSF

List of Acronyms
and Abbreviations17
References���������� 17

FIGURE 1.1 RASSF protein structure.

Protein structures for the C-terminal RASSF members are shown. C1, zinc finger domain; RA, Ras
association domain; SARAH, Salvador/RASSF/Hippo domain.

extensive epigenetic inactivation in numerous cancers, thus they will be the

focus of this discussion.
A general feature of the RASSF proteins is that they do not appear to have any
enzymatic activity; instead, they appear to act as scaffolding molecules, facil-
itating the growth and survival suppressing effects of Ras by scaffolding it to
various pro-apoptotic or pro-senescent signaling pathway proteins. All of the
C-terminal RASSF proteins also contain a Salvador/RASSF/Hippo, or SARAH,
domain, which directly binds the mammalian sterile 20 like (MST) kinases,
connecting them to the Hippo signaling pathway [6,7]. Relevant structural
domains of the C-terminal RASSF proteins are highlighted in Fig. 1.1.
Another unique feature of RASSF proteins is their high rate of epigenetic inactiva-
tion in numerous cancers. Epigenetics refers to changes in gene expression that are
not due to changes in the DNA sequence itself, and in the case of RASSF proteins,
they commonly experience methylation of CpG islands in their promoter regions,
leading to the loss of expression of RASSF proteins in the cell. Suppressing RASSF
proteins experimentally can enhance Ras transformation and disconnect Ras from
apoptotic and senescent pathways [8,9]. Thus loss of RASSF protein expression
facilitates Ras transformation. The known relevance of each of the C-terminal
RASSF proteins to Ras function will be summarized in the following sections.

RAS AND RASSF1

The RASSF1 gene was identified serendipitously in a two-hybrid screen for pro-
teins that interact with the DNA repair protein xeroderma pigmentosum group
A-complementing protein (XPA) [10]. It was shown to produce two main
 Ras and RASSF1 5

transcripts, RASSF1A and RASSF1C, both of which contain an RA domain. Sev-

eral other isoforms appear to exist, but because there is little or no data avail-
able on them, they will not be considered here.
The RASSF1 proteins were shown to bind to activated Ras and promote Ras-
dependent apoptosis [9,11]. Initially, some controversy arose as to the physio-
logical nature of the interaction between Ras and RASSF1A, with some groups
suggesting the interaction did not occur or was indirect [12]. However, multi-
ple groups have now reported that activated Ras forms an endogenous complex
with RASSF1A and that the interaction is likely to be direct [9,11,13]. A possible
explanation for the confusion was identified when we observed that RASSF1A
preferentially associates with K-Ras and fails to bind non-farnesylated Ras [2].
Consequently, experiments using recombinant H-Ras protein from bacteria or
non-farnesylated Ras mutants in yeast would not be expected to give positive
results.
Early work showed that RASSF1A was frequently down-regulated in human
tumor cells and could act to suppress the tumorigenic phenotype in vitro
[9,10]. RASSF1A has no apparent enzymatic activity, and we hypothesize that
it acts as a scaffolding protein under the control of K-Ras. This allows K-Ras to
control multiple tumor suppressing pathways.
The first biological properties of RASSF1A that were characterized were that
RASSF1A can promote both G1 and G2/M cell cycle arrest [6,9,14]. The G2/M
arrest can be explained by the powerful effects of RASSF1A over-expression on
microtubule polymerization. RASSF1A directly binds multiple microtubule-as-
sociated proteins (Maps), which themselves directly bind to tubulin [15,16].
Maps modulate microtubule polymerization. We have found RASSF1A asso-
ciating with most forms of tubulin, including gamma tubulin at the spindle
poles, and this may explain the ability of RASSF1A to suppress K-Ras-induced
genetic instability [16]. A more technically sophisticated study suggested that
in interphase cells, RASSF1A preferentially associates with a subset of micro-
tubules at the Golgi to promote correct cell polarity and Golgi orientation
[17]. In addition to a microtubular localization, we can identify endogenous
RASSF1A in the nuclear compartment, and the protein has also been reported
to associate with mitochondria [18].
In addition to its effects on microtubules, RASSF1A has also been shown to
connect Ras to two major pro-apoptotic signaling pathways: the Bax pathway
and the Hippo pathway. Bax is a pro-apoptotic protein that contains a Bcl-2
homology, or BH, domain and is critical for most forms of apoptosis in the
cell. In 2005, two studies by independent groups identified RASSF1A as a crit-
ical mediator of Bax activation through its ability to directly interact with the
protein modulator of apoptosis-1 (MOAP-1) [19,20]. MOAP-1 in turn directly
binds and activates Bax. Activated K-Ras enhances the interaction of RASSF1A
6 CHAPTER 1: Ras and RASSF

and MOAP-1 to stimulate Bax activation and translocation to the mitochon-

dria. Suppressing RASSF1A impairs the ability of Ras to activate Bax in tumor
cells [20].
RASSF1A also connects Ras to another major pro-apoptotic signaling path-
way, the Hippo pathway. The major splice variants of RASSF proteins 1–6 all
contain a C-terminal SARAH motif. This serves to bind to the Hippo kinases
MST1 and MST2 [6,7,11,21]. The MST kinases can in turn phosphorylate and
activate the large tumor suppressors (LATs) kinases in a kinase cascade. LATs
kinases have several targets, but the most important targets appear to be the
transcriptional co-activators yes-associated protein (YAP) and tafazzin (TAZ)
[22]. Phosphorylation of YAP/TAZ by LATs promotes their exclusion from the
nucleus and leads to their proteosomal degradation. YAP acts as a potent onco-
gene and pro-survival factor, so its suppression by the Hippo pathway can lead
to apoptosis or senescence [22]. The Hippo pathway plays a key role in normal
cellular homeostasis, and it is commonly dysregulated in human cancers, lead-
ing to YAP activation and pro-growth effects (reviewed in Ref. [23]). RASSF1A
serves to connect Ras to the control of the Hippo pathway as the interaction of
Ras with RASSF1A promotes MST kinase stability and activation [24,25]. Thus
the loss of RASSF1A uncouples Ras from the activation of the Hippo pathway,
suppressing apoptotic signaling. However, this story may be even more com-
plex. In an in vivo system using a point mutant of RASSF1A that specifically
fails to bind the MST kinases, we found that cardiomyocytes and cardiac fibro-
blasts react quite differently to RASSF1A/Hippo signaling [24]. Thus there may
be a strong cell type specificity associated with the net result of this pathway.
RASSF1A has also been shown to complex with the mouse double minute
2 homolog (MDM2) ubiquitin ligase, which can degrade both p53 and Rb
[26]. By promoting the degradation of MDM2, RASSF1A may stabilize p53 and
potentially Rb, thereby activating them. This connection with p53 may explain
why RASSF1A/p53 dual heterozygous knockout mice exhibit synergistic tumor
formation [27]. The role of Ras in this process remains unclear.
Furthermore, RASSF1A has now been shown to play an important role in both
the DNA damage response and the DNA repair process itself [28–30]. The
O’Neill group showed that RASSF1A is involved in activating MST2 and LATS1
upon DNA damage, leading to the stabilization of the pro-apoptotic protein
p73 [29]. Therefore, RASSF1A-defective cells fail to activate apoptotic processes
when they are subjected to DNA damage, thus promoting the survival of cells
carrying mutations, which can lead to cancer.
Donninger et al. showed that RASSF1A-defective cells not only fail to induce
the DNA damage apoptotic response, but also fail to repair that DNA dam-
age. They found that the original yeast two-hybrid observation that RASSF1A
might bind the DNA repair protein XPA was in fact correct [31]. Moreover, they
 Ras and RASSF1 7

found that RASSF1A-negative cells were defective for proper XPA regulation
and, as a result, were less able to repair DNA damage due to UV radiation. This
observation was confirmed in vivo by enhanced tumor formation in UV-ir-
radiated mice heterozygous for both RASSF1A and XPA [31]. Intriguingly, a
single nucleotide polymorphism (SNP) variant of RASSF1A that exhibits an
alteration in the consensus phosphorylation site for the DNA damage kinases
ataxia telangiectasia mutated protein/ataxia telangiectasia and Rad3-related
protein has been identified. This variant was found to be defective for both the
DNA damage response and for supporting DNA repair after damage [29,31].
Thus SNP carriers are both less able to respond to DNA damage by inducing
cell death and less able to repair DNA damage. These observations explain the
reported enhanced cancer predisposition of SNP carriers but fail to explain
why the SNP is so common in European populations (∼22%) but very rare in
African populations (∼2%) [32–34]. We speculate that the attenuated apop-
totic properties of the SNP variant may give some biological advantage to car-
riers. RASSF1A has been shown to play an important role in cell death during
cardiac hypertrophy [35]. Perhaps, the attenuated apoptotic response due to
the SNP variant may suppress cardiac disease.
Further investigation showed us that the mechanism by which RASSF1A
appears to control DNA repair involves regulating the acetylation status of
DNA repair proteins via the SIRT1 deacetylase [31]. It has been reported that
RASSF1A can form a complex with the deacetylase HDAC6 [36]. Thus RASSF1A
can link K-Ras to the control of protein acetylation by multiple deacetylases.
Loss of RASSF1A may induce general defects in the acetylome. As acetylation
may be an even more widespread post-translational modification in the cell
than phosphorylation [37], this effect could be of profound importance to
Ras-driven tumor development and to tumor response to acetyl transferase
inhibitors.
Transgenic mouse studies have confirmed a tumor suppressor role for RASSF1A
in vivo as knockout mice developed a modest increase in spontaneous tumor
development with age or carcinogen treatment [38]. However, the results were
subtle and curious in that the heterozygous knockout mice developed more
tumors than the homozygous knockout mice. This hints that the cell may
require a minimal, reduced RASSF1A expression for survival. Indeed, potent
suppression of many tumor suppressors, such as breast cancer early onset 1 or
von hippel-lindau, can lead to the reduced growth of target cells, and the same
appears to be true for RASSF1A [39]. This may explain why RASSF1A is so sel-
dom completely deleted in human cancer. Studies examining the loss of both
RASSF1A and p53 showed a synergistic effect, with RASSF1A-null, p53-null
mice showing a large amount of spontaneous tumor formation at a young age
[27]. It will be revealing to examine the results of RASSF1A suppression and
Ras activation in mouse models.
8 CHAPTER 1: Ras and RASSF

The main alternative splice form of RASSF1 is RASSF1C, a shorter form that
lacks the N-terminus of RASSF1A. RASSF1C can complex with K-Ras and has
apoptotic properties [9]. The RASSF1C promoter has not been reported to suf-
fer epigenetic inactivation in tumors, so the protein is not regarded as a tumor
suppressor. However, we have found that RASSF1C protein expression is lost
in some tumor cell lines. Indeed, it is lost in some cases where the RASSF1A
protein expression is retained (unpublished data). This implies that RASSF1C
may be regulated at a post-transcriptional level and may also act as a tumor
suppressor, at least in some cell types.
Contradictory roles for RASSF1C have also been reported. In our hands, it
appears to behave rather like a weaker form of RASSF1A, polymerizing micro-
tubules and promoting apoptotic cell death [16]. It has also been shown to
play a role in ovarian cancer cell death and in the activation of the apoptotic
jun N-terminal kinase (JNK) pathway after DNA damage [40,41]. Other groups
have found that RASSF1C can have a mild stimulatory effect on tumor cell
growth and may up-regulate the β-catenin oncoprotein [42–44]. Consequently,
the physiological functions of this isoform remain unclear.

RAS AND RASSF5 (NORE1)

The second best-studied RASSF family member is RASSF5. The RASSF5 gene
produces two main protein isoforms, RASSF5A, also known as NORE1A, and
NORE1B, or RAPL. NORE1A is broadly expressed in tissue, whereas NORE1B
seems mostly restricted to the lymphoid compartment. NORE1A was origi-
nally identified as a Ras-binding protein in a two-hybrid screen [45]. RAPL/
NORE1B was identified as a Rap binder in a similar screen. NORE1A binds to
Ras via the effector domain in a GTP-dependent manner [46]. It can be found
in an endogenous complex with Ras, so it meets the definition of a Ras effector.
Unlike RASSF1A, it readily binds H-Ras [2].
NORE1A is often down-regulated in tumors by epigenetic mechanisms [2]. It
can also be down-regulated at a protein level in tumor cells by calpains and by
ubiquitination [47,48]. In liver cancer, more malignant primary tumor samples
expressed less NORE1A [13]. Moreover, a human family with a translocation
that inactivates the NORE1A gene suffers from a hereditary cancer syndrome
[49]. Thus the evidence that NORE1A is a tumor suppressor is strong.
Ras can use NORE1A as a pro-apoptotic effector [7]. Like RASSF1A, NORE1A
binds the MST kinases and has the potential to modulate the pro-apoptotic
Hippo pathway. However, deletion mutagenesis has shown that the canonical
Hippo pathway is not essential to the growth suppressing function of NORE1A,
and it is unclear if NORE1A can stimulate the canonical Hippo kinase cascade
[21,50].
 Ras and RASSF5 (NORE1) 9

We find that NORE1A is a highly potent senescence effector of Ras [8]. Over-
expression of NORE1A induces senescence at levels comparable with those
induced by activated Ras, whereas suppression of NORE1A severely impairs the
senescence response and enhances Ras-mediated transformation [8]. Moreover,
the expression of NORE1A in primary human tumors correlates well with the
expression of senescence markers [51]. Specifically, NORE1A can form a Ras-­
regulated complex with p53 and scaffolds it to the kinase HIPK2, so in the
presence of NORE1A, we can detect Ras in a complex with p53 [8]. Thus the
long-known but poorly understood link between Ras and p53 may be solved by
NORE1A. HIPK2 can phosphorylate p53 at residue S46 to promote apoptosis;
however, it can also recruit acetyl transferases to acetylate p53 to promote its
pro-senescence effects (reviewed in Ref. [52]). NORE1A acts to suppress S46
phosphorylation of p53 and enhances p53 acetylation at residues 382 and 320,
thus driving p53-dependent senescence [8]. Once again, this is evidence that
RASSF proteins may be able to couple Ras to the control of protein acetylation.
In addition, NORE1A can associate with MDM2, a negative regulator of p53,
but it can use this association to induce the ubiquitination and degradation of
the oncoprotein HIPK1 by scaffolding the two proteins together, highlighting
the functionality of NORE1A as a scaffolding molecule [53]. In our hands, the
NORE1A/MDM2 interaction appears to be Ras regulated, adding a further level
to the Ras/NORE1A/p53 relationship.
Another major component of Ras/NORE1A signaling that has been identified
is the β-catenin protein [54]. β-catenin is an adherens junction protein and
transcription co-factor that serves as the terminal executor of the Wnt signaling
pathway. A multi-protein complex normally phosphorylates β-catenin, allow-
ing it to bind the SCFβ-TrCP ubiquitin ligase complex [55]. In the absence
of Wnt stimulation, this process results in β-catenin degradation. When Wnt
is activated, un-phosphorylated β-catenin translocates to the nucleus, where
it activates the transcription of growth-promoting, pro-survival genes. Thus
β-catenin, when either dysregulated or mutated, can act as an oncogene in
cancer, and β-TrCP, the SCFβ-TrCP substrate recognition component, can act as
a tumor suppressor because of its influence on β-catenin degradation. Schmidt
et al. found that NORE1A plays a role in this signaling process by directly bind-
ing β-TrCP in an interaction that is enhanced by activated Ras, allowing Ras
to specifically stimulate SCFβ-TrCP-mediated degradation of β-catenin [54].
Thus in cancers where NORE1A expression is lost, negative Ras regulation of
β-catenin is disrupted, revealing another crucial barrier that the RASSF proteins
present to uncontrolled cell growth and tumorigenesis [54].
In vivo studies investigating the role of NORE1A as a potential tumor suppressor
found that NORE1A knockout mice appear overtly normal, yet mouse embry-
onic fibroblasts (MEFs) from the animal can be transformed by activated Ras
10 CHAPTER 1: Ras and RASSF

in a single step [56]. Normal MEFs require the addition of other oncogenic
events such as mutant p53 or the activation of other oncogenes to suppress
oncogene-induced senescence to transform [5], so the loss of NORE1A increases
the susceptibility to transformation. In addition, the same study revealed that
NORE1A helps mediate tumor necrosis factor (TNF)-α–mediated, or death
receptor mediated, apoptosis, most likely through its interaction with MST1,
providing further evidence that NORE1A is a tumor suppressor [56]. In human
tumors, few studies have examined the results of NORE1A inactivation and Ras
activation, but one study of hepatocellular carcinomas with activated Ras signal-
ing found that NORE1A promoter methylation was only found in a subset of
tumors with poor patient survival, indicating the potential impact of NORE1A
loss on human tumors [13]. Studies of NORE1A-null, Ras-positive mice will pro-
vide more insight into the full inhibitory effect that NORE1A plays in cancer.
A lesser-studied NORE1 isoform, NORE1B has mostly been shown to play
a role in immune cells, and it seems to be expressed more in the lymphoid
compartment compared with the fairly ubiquitous expression of other RASSF
proteins. Specifically, NORE1B plays an important role in lymphocyte and
dendritic cell adhesion and migration and was shown to be a crucial part
of immunosurveillance. The loss of NORE1B expression in a mouse model
resulted in immune dysfunction that included the inability of lymphocytes
and dendritic cells to migrate to tissues and impaired the maturation of B cells
[57]. Like NORE1A, NORE1B can associate with MST1, and the two form a
synergistic relationship, negatively regulating T cell proliferation when the T
cell antigen receptor is stimulated [58]. In addition, NORE1B has been shown
to work with Ras to regulate T cell signaling; NORE1B recruits activated Ras to
T cell synapses to promote Ras signaling in immune cells [59].
Unlike the other C-terminal RASSF proteins, very little evidence exists to show
that NORE1B can act as a tumor suppressor or that it experiences epigenetic inac-
tivation, but it can associate with Ras and Ras-related proteins [60]. One study
revealed that NORE1B experiences a high percentage (62%) of promoter meth-
ylation in hepatocellular carcinoma, indicating that NORE1B loss may enhance
tumorigenesis in some systems [61]. The same group found that NORE1B inter-
acts with RASSF1A and that the two are frequently lost together in hepatocellular
carcinomas, leading to the hypothesis that NORE1B and RASSF1A work together
to prevent hepatocellular carcinoma formation [62]. Further work on this sub-
ject is needed to elucidate the mechanism of action of NORE1B.

RAS AND RASSF2

RASSF2 forms an endogenous complex with activated K-Ras via the Ras effec-
tor domain. It appears to be specific for K-Ras as its interaction with H-Ras is
very weak [63]. RASSF2 is expressed at particularly high levels in the brain but
 Ras and RASSF3 11

is expressed in many other tissues at lower levels. Like other RASSF proteins,
RASSF2 has been found to undergo epigenetic inactivation in a large number
of cancers, and its inactivation was found to increase oncogenic transforma-
tion induced by K-Ras in colorectal cancer and in lung cancer cells [64,65].
RASSF2 has also been identified as a potential metastasis suppressor [66]. Loss
of RASSF2 expression enhances cell growth, disrupts adhesion, and leads to the
up-regulation of phosphorylated AKT [65]. Curiously, RASSF2 knockout mice
die soon after birth. Thus RASSF2 may actually be the most critical member of
the RASSF family as it is the only one that is essential to life.
Of all the tumor types screened, RASSF2 shows the most intense level of aber-
rant promoter methylation in prostate tumors, with up to 95% promoter
methylation observed in one study [67]. Moreover, this methylation correlated
well with the frequent loss of protein expression in primary prostate cancers.
RASSF2 may prove to be a highly effective diagnostic marker for prostate can-
cer, as it is so commonly methylated, and RASSF2 promoter methylated DNA
can be detected in urine samples by a sensitive polymerase chain reaction assay
[68]. Indeed, this was found to be a more predictive biomarker for prostate
cancer than the prostate-specific antigen test. RASSF2 has also been proposed
as a potential biomarker for gliomas [69].
RASSF2 is pro-apoptotic and acts, in part, by binding the prostate apoptosis
response protein (PAR-4). This interaction is K-Ras regulated, and activated
K-Ras promotes translocation of PAR-4 to the nucleus via RASSF2. There,
PAR-4 can interact with the TNF-related apoptosis-inducing ligand to induce
apoptosis [67]. RASSF2 can also bind the MST kinases and may modulate their
stability. However, like NORE1A, RASSF2 can induce apoptosis independently
of MST [70,71]. RASSF2 can also modulate both nuclear factor κb signaling
and the JNK pathway, but the precise mechanisms of these effects and if they
are coupled to Ras remain to be elucidated [70,72].

RAS AND RASSF3

We have found that RASSF3 can bind activated K-Ras in over-expression systems,
but an endogenous complex between Ras and RASSF3 has yet to be confirmed.
We also found that K-Ras and RASSF3 co-operate to induce cell death. We have
not found much evidence of RASSF3 methylation or protein down-regulation in
tumors in our studies; however, RASSF3 may be deleted in some colorectal can-
cers and in patients with relapsed acute lymphoblastic leukemia [73,74]. More-
over, several RASSF3 SNPs have been reported to be associated with an enhanced
risk of head and neck cancer [75]. In one tumor type, somatotroph adenomas,
high-frequency promoter methylation has been reported [76]. Thus, although
there is evidence for RASSF3 loss of function in human cancer, this is a much less
frequent event than that observed for some other family members.
12 CHAPTER 1: Ras and RASSF

Experimentally, RASSF3 inactivation has been reported to lead to defects in

DNA repair and enhanced genomic instability as well as enhanced transforma-
tion of lung tumor systems [77,78]. RASSF3 can also bind MST1 but does not
appear to activate it [7]. Instead, RASSF3 is involved in p53-mediated apopto-
sis and has been shown to modulate p53 via binding and promoting MDM2
degradation [76,78].

RAS AND RASSF4 (AD037)

RASSF4 (originally designated AD037) can bind activated K-Ras via the effec-
tor domain [79], but the lack of good antibodies for RASSF4 has prevented
the confirmation of endogenous complex formation between the two. Over-
expressed RASSF4 promotes a Ras-dependent apoptosis, and RASSF4 expression
is down-regulated by promoter methylation in some tumor cells, including
nasopharyngeal carcinoma [79,80]. RASSF4 down-regulation has also been
linked to the maintenance of cancer stem cells in oral squamous cell cancer
stem-like cells [81].
Although RASSF4 is down-regulated in some tumors, we did observe that some
primary human breast cancers exhibited enhanced RASSF4 expression [79].
Moreover, RASSF4 has been implicated as pro-oncogenic by binding to MST1
and inhibiting the Hippo pathway in alveolar rhabdomyosarcoma systems,
resulting in increased YAP expression and increased cell growth and senescence
evasion [82]. Thus RASSF4 may have different biological effects in different cell
systems.

RAS AND RASSF6

RASSF6 can bind activated K-Ras via the effector domain in over-expression
studies but has yet to be confirmed in an endogenous complex with Ras.
RASSF6 can induce apoptosis, and suppression of RASSF6 can enhance the
transformed phenotype of tumor cell lines [83,84]. RASSF6 is epigenetically
down-regulated in primary cancers although less so than RASSF1A or RASSF5.
It has been found to be specifically inactivated in neuroblastoma, childhood
leukemias, and melanoma and melanoma metastases [85–87].
RASSF6 was the first RASSF family member to be identified that binds and inhib-
its, rather than activates, the MST kinases to suppress the Hippo pathway [88].
This is the same effect as observed with the single Drosophila RASSF protein.
Therefore, RASSF6 must induce apoptosis independently of Hippo signaling. As
with other RASSF family members, RASSF6 interacts with the MDM2 protein
to modulate p53, apoptosis, and the cell cycle [89]. Like RASSF1A, RASSF6 can
form a Ras-regulated complex with the Bax activating protein MOAP-1 [83,84].
In addition, over-expressed RASSF6 has been shown to increase the association
 Therapeutic Ramifications 13

of the inhibitory kinase MST1 and mutant B-Raf to suppress the mitogen acti-
vated protein kinase (MAPK) pathway in melanoma cells [87]. Thus RASSF6
has multiple mechanisms that could be used by Ras to suppress tumorigenesis.
However, we have observed occasional strong up-regulation of RASSF6 in some
primary tumor samples, such as ovarian. Perhaps, like the Hippo-inhibiting
RASSF4 protein, in some circumstances, RASSF6 may be pro-tumorigenic.

EFFECTS OF RASSF PROTEINS ON MITOGENIC RAS

EFFECTORS
RASSF proteins bind Ras and can be considered as Ras death effectors con-
necting Ras to multiple signaling pathways that can mediate apoptosis or
senescence. However, the role of RASSF proteins in Ras biology may be more
complex and subtle. In addition to their own signaling pathways, RASSF pro-
teins may be able to modulate the activity of the classic mitogenic signaling
pathways used by Ras.
The MST2 kinase not only binds RASSF1A but it can also bind Raf-1, where
it serves to inhibit Raf kinase activity [25,90]. RASSF1A acts to compete with
Raf-1 for MST2 binding, so down-regulating RASSF1A can increase Raf-1/
MST2 binding, suppressing the MAPK pathway. Thus Ras modulates Raf-1
directly by binding to it and indirectly via RASSF1A/MST2. RASSF1A has also
been reported to suppress AKT activity by a mechanism that remains unclear
[91]. AKT is a component of the Ras/phosphoinositide 3-kinase pathway. In a
further twist, AKT can phosphorylate MST2 to promote its binding to Raf-1,
inhibiting the kinase activity of MST2 [11,25]. We have found that RASSF6 can
also modulate the interaction of MST1 with activated B-Raf in a melanoma
cell line to suppress the MAPK pathway [87]. Thus RASSF proteins may have
a more complex role in mediating Ras biology than simply controlling their
own, separate signaling modalities. They may be able to integrate the regula-
tion of pro-mitotic and pro-death Ras pathways.

THERAPEUTIC RAMIFICATIONS
RASSF1A is epigenetically inactivated in a broad range of primary human
tumors [92,93]. For reviews of cancers known to experience epigenetic inac-
tivation of RASSF1A, see Refs. [2,94]. Loss of RASSF1A uncouples Ras from
multiple growth suppressive pathways, so it would seem reasonable that Ras
tumors would often show RASSF1A down-regulation.
Although some studies have shown a correlation between Ras point mutations
and RASSF1A promoter methylation, the majority have not. However, in addition
to inactivation by promoter methylation, RASSF1A can be inactivated by point
14 CHAPTER 1: Ras and RASSF

mutations at a significant frequency [94,95]. Moreover, a SNP variant of RASSF1A

has been identified that is defective for some apoptotic responses and predisposes
carriers to cancer development [32,33]. Thus many RASSF1A “positive tumors,” as
measured by promoter methylation, may actually be RASSF1A negative. Moreover,
Ras is frequently activated in the absence of point mutations by defects in upstream
activators (eg, human epidermal growth factor receptor 2) or negative regulators
such as neurofibromatosis 1, Ras GTPase activating like protein, or DAB2 interact-
ing protein [96–98]. Therefore, assays performed at a protein level will be required
to definitively answer the question of the relationship between Ras activation and
RASSF inactivation in human tumorigenesis.
One large-scale study that stratified non-small cell lung cancer tumors and mea-
sured Ras mutation and RASSF1A promoter methylation has been reported [99].
It showed that, although there was no general correlation, tumors with both
K-Ras mutations and RASSF1A methylation had a much poorer prognosis and
lower overall patient survival than other tumors of the same stage [99]. A simi-
lar result was reported for hepatocellular carcinoma tumors with NORE1A pro-
moter methylation and Ras activation [13]. This implies that most tumors with
Ras activation and RASSF methylation have the potential to be more aggressive
than tumors without RASSF methylation, data that certainly correlate well with
studies examining the loss of RASSF proteins in K-Ras–positive cancer cell lines.
For example, the loss of RASSF2 expression was shown to enhance proliferation
and invasion of K-Ras–positive lung cancer cells, and it also conferred resistance
to chemotherapy in those cells [65]. Similar results were obtained for RASSF3 in
non-small cell lung cancer, and our group showed that reintroduction of RASSF6
expression in a metastatic melanoma cell line that had lost RASSF6 expression
was sufficient to alter mutant B-Raf signaling and decrease the invasiveness of
those cells [77,87]. In addition, RASSF1A knockdown cells exhibit resistance
to DNA damage-induced apoptosis and to treatment with cisplatin [29]. These
observations suggest that epigenetic therapy designed to restore RASSF protein
expression might be a plausible strategy to help treat aggressive Ras-positive,
RASSF-negative tumors. This might be a particularly attractive approach as tar-
geting Ras directly has so far not been successful [100].
DNA methylation is a reversible process, making it a potential target for cancer
therapy. DNA is methylated by DNA methyltransferase (DNMT) proteins, and
a class of drugs called DNA methyltransferase inhibitors can be used to prevent
DNMTs from methylating DNA. The most commonly used DNA methyltransfer-
ase inhibitors are 5-azacytidine and decitabine, both of which are nucleoside ana-
logs that cause DNA methyltransferases to be inactivated in a protein–DNA adduct
[101]. Both these drugs have been approved for the treatment of myelodysplastic
syndrome and for low-blast count acute myeloid leukemia, but their efficacy in
the treatment of solid tumors is limited, potentially because of the higher doses
needed that lead to unwanted side effects, like myelosuppression and nausea [102].
 Conclusion 15

The aberrant methylation of the RASSF1A promoter appears to be mediated

primarily by one enzyme: the DNA methyltransferase DNMT3B [103]. This
protein can be up-regulated by K-Ras, so it is possible that Ras mutations
actually promote RASSF1A inactivation [104]. A quinone-based antibiotic,
nanaomycin A, was identified as a specific DNMT3B inhibitor. Nanaomycin
A treatment was able to result in specific re-expression of RASSF1A in lung
cancer cells, and the tumorigenic phenotype in those cells was effectively
suppressed [105]. Similar results were also shown in a melanoma cell line,
in which treatment with nanaomycin A resulted in re-expression of RASSF6
[87]. Nanaomycin A has also been shown to eradicate melanoma stem cell–
like cancer cells [106]. These results imply that nanaomycin A treatment,
or drugs with a similar action, has considerable potential to result in re-
expression of all RASSF proteins in Ras-driven tumor cells. Such a treatment
might have therapeutic potential in many different cancers without the side
effects associated with the use of less specific nucleoside analogs. No clin-
ical trials that examine the utility of nanaomycin A as an anti-cancer agent
have currently been reported.
Another aspect of clinical relevance for RASSF proteins is their use as biomark-
ers in human tumors. RASSF1A down-regulation, thought to be one of the
most common events in human cancer, is widely associated with more aggres-
sive cancer phenotypes, and methylation of RASSF1A DNA promoter regions
can be detected in sputum, serum, and urine analyses [93]. RASSF2 methyl-
ation can also be detected in the urine of patients with prostate cancer [68].
Overall, examining the methylation status of RASSF proteins is a non-invasive
process that could provide insight into the overall cancer phenotype, and this
information could be used to develop a more personalized treatment plan for
patients with cancer.
In addition, an RASSF1A SNP variant has been discovered that renders cells
less sensitive to cell death by DNA damaging agents [29]. Patients with this
polymorphism could thus experience different responses to certain chemo-
therapeutic regimens, meaning that not only RASSF1A expression status but
also RASSF1A mutation status could play a role in developing personalized
therapies for patients with cancer.

CONCLUSION
Ras activation is likely to be the most common single event in the develop-
ment of human cancer. When the proper checkpoints are in place, however,
Ras activation does not lead to cancer. Instead, activated Ras can promote cell
death and/or cell growth arrest. This surprising function of a widely known
oncogene is facilitated by the RASSF protein family. Members of this family
have no enzymatic activity and instead work by scaffolding Ras to various
16 CHAPTER 1: Ras and RASSF

Ras

RASSF1A
RASSF1A
PI3K
RAF
MST
1/2 MOAP-1 MDM2

AKT
MEK LATS Bax p53
1/2

MAPK
YAP/TAZ Cell cycle
arrest

Proliferation/ Apoptosis
transformation Senescence
FIGURE 1.2 Signaling pathway involvement of RASSF1A.
RASSF1A is involved in a wide variety of signaling pathways, several of which are outlined here. RASSF1A
can act to stimulate apoptosis upon Ras activation, but it can also act independently of Ras to inhibit Akt
signaling through several mechanisms described in this discussion.

pro-apoptotic and pro-senescent signaling pathways. In addition, RASSF

family members can impact the activity of other mitogenic Ras effectors or
effector pathways. A summary of RASSF protein involvement in Ras signaling
pathways is shown in Fig. 1.2, using RASSF1A as an example to show the
wide variety of processes in which RASSF signaling plays a major role.
Further studies are needed to elucidate the full significance of losing RASSF pro-
teins in Ras-driven tumors. For example, in the case of RASSF1A, RASSF1A-null
mice show an increased susceptibility to tumors, yet the dual effect of RASSF1A
loss and Ras activation remains to be examined [38]. Therapeutic approaches
aimed at reactivating the expression of these proteins in cancer have the poten-
tial to offer novel, personalized treatments for patients with RASSF promoter
methylation. Overall, RASSF proteins showcase an interesting and paradoxical
side of Ras, and their potential clinical utility could provide a useful tool for
targeting a large subset of the most intractable Ras-driven tumors in the future.

Glossary
Apoptosis Programmed cell death.
Epigenetics The study of changes that are caused by the modification of gene expression, not by
alteration of DNA itself.
Kinase An enzyme that catalyzes the transfer of a phosphate group from a molecule of ATP to
another molecule.
Methylation The addition of a methyl group (CH3) to a DNA base. Methylation of cytosines in
CpG island regions of DNA can lead to gene suppression.
 References 17

Mitogenic A growth-promoting (mitosis-inducing) substance or signal.

Senescence A state of permanent cell cycle arrest, in which the cell is alive but not actively growing.

List of Acronyms and Abbreviations

ALL Acute lymphoblastic leukemia
ATM/ATR Ataxia telangiectasia mutated protein/ataxia telangiectasia and Rad3-related protein
BRCA1 Breast cancer early onset 1
DAB2IP DAB2 interacting protein
DNMT DNA methyltransferase
GAPs GTPase-activating proteins
HDAC6 Histone deacetylase protein 6
HER2 Human epidermal growth factor receptor 2
JNK Jun N-terminal kinase
LAT Large tumor suppressor kinase
MAPK Mitogen activated protein kinase
Maps Microtubule-associated proteins
MDM2 Mouse double minute 2 homolog
MOAP-1 Modulator of apoptosis-1
MST Mammalian sterile 20 like
NF1 Neurofibromatosis 1
PAR-4 Prostate apoptosis response protein 4
RA domain Ras-association domain
RASAL Ras GTPase activating like protein
RASSF Ras-association domain family
SARAH domain Salvador/RASSF/Hippo domain
SIRT1 NAD-dependent protein deacetylase sirtuin-1
TAZ Tafazzin
TRAIL TNF-related apoptosis-inducing ligand
VHL Von hippel-lindau
XPA Xeroderma pigmentosum group A-complementing protein
YAP Yes-associated protein

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