Advances in Biological Regulation 55 (2014) 114

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Advances in Biological Regulation

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

An expanding role for RAS GTPase activating

proteins (RAS GAPs) in cancer
Ophlia Maertens a, b, Karen Cichowski a, b, c, *
a
Genetics Division, Department of Medicine, Brigham and Womens Hospital, Boston, MA 02115, USA
b
Harvard Medical School, Boston, MA 02115, USA
c
Ludwig Center at Dana-Farber/Harvard Cancer Center, Boston, MA 02115, USA

a b s t r a c t

Keywords: The RAS pathway is one of the most commonly deregulated

RAS pathways in human cancer. Mutations in RAS genes occur in nearly
GAP 30% of all human tumors. However in some tumor types RAS
GTPase activating protein
mutations are conspicuously absent or rare, despite the fact that
RAS GAP
RAS and downstream effector pathways are hyperactivated.
NF1
RASAL2 Recently, RAS GTPase Activating Proteins (RAS GAPs) have
DAB2IP emerged as an expanding class of tumor suppressors that, when
IQGAP inactivated, provide an alternative mechanism of activating RAS.
Therapeutic resistance RAS GAPs normally turn off RAS by catalyzing the hydrolysis of
RAS-GTP. As such, the loss of a RAS GAP would be expected to
promote excessive RAS activation. Indeed, this is the case for the
NF1 gene, which plays an established role in a familial tumor
predisposition syndrome and a variety of sporadic cancers. How-
ever, there are 13 additional RAS GAP family members in the
human genome. We are only now beginning to understand why
there are so many RAS GAPs, how they differentially function, and
what their potential role(s) in human cancer are. This review will
focus on our current understanding of RAS GAPs in human disease
and will highlight important outstanding questions.
2014 Elsevier Ltd. All rights reserved.

* Corresponding author. 458d NRB, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Tel.: 1 (617) 525 4722.
E-mail address: kcichowski@rics.bwh.harvard.edu (K. Cichowski).

http://dx.doi.org/10.1016/j.jbior.2014.04.002
2212-4926/ 2014 Elsevier Ltd. All rights reserved.
2 O. Maertens, K. Cichowski / Advances in Biological Regulation 55 (2014) 114

Introduction

RAS proteins are small molecular weight GTPases that couple extracellular signals to intracellular
effector pathways. The most extensively studied RAS isoforms (H-RAS, K-RAS and N-RAS) play a critical
role in fundamental cellular processes, including proliferation, survival, differentiation, motility and
transcription (Karnoub and Weinberg, 2008). RAS proteins together with their two key regulators,
guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), constitute mo-
lecular switches that cycle between on and off conformations caused by the binding of GTP or GDP,
respectively (Fig. 1). The transition between the inactive GDP-bound and active GTP-bound state of RAS
is controlled by GEFs, which activate RAS by promoting the exchange of GDP for GTP, and GAPs, which
turn off RAS by catalyzing RAS-mediated GTP hydrolysis (Bos et al., 2007). The RAS pathway is one of
the most commonly deregulated pathways in human cancer (Karnoub and Weinberg, 2008). Activating
mutations in RAS genes occur in many different tumor types. These mutations typically render RAS
constitutively GTP-bound, resulting in the activation of downstream effector pathways in the absence
of extracellular stimuli (Karnoub and Weinberg, 2008). However, RAS GAPs have emerged as an
expanding new class of tumor suppressor genes. Accumulating evidence indicates that inactivation of
several RAS GAP family members represents an important and alternative mechanism to (hyper)
activate RAS and its downstream effectors in tumors (Fig. 1). This review will focus on the emerging
biological role of RAS GAPs in human cancer.
There are 14 predicted RAS GAP genes in the human genome (Bernards, 2003). All contain a RAS
GAP domain but share little similarity outside of this region (Fig. 2). Interestingly, members of the
IQGAP subfamily contain an alternative amino acid within a key region of the catalytic domain, and
therefore do not exhibit RAS GAP activity but still affect RAS signaling (Brill et al., 1996; Wang et al.,
2007; Weissbach et al., 1994). Regions anking the RAS GAP domains are thought to promote pro-
teinprotein and proteinlipid interactions, second messenger binding and phosphorylation by protein
kinases. These interactions may facilitate association with specic subcellular membranes or com-
partments, regulating spatially restricted GTPase activation (Bernards and Settleman, 2004). Accord-
ingly, individual RAS GAPs may target specic GTPases by associating with specic membranes and/or
signaling complexes. Furthermore, RAS GAP activity and its effects on downstream signaling pathways
appears to be regulated in response to specic growth factors which is likely tissue- and context-
specic (Bernards and Settleman, 2005; Cichowski et al., 2003; McGillicuddy et al., 2009). Finally, at
least some RAS GAPs also have distinct RAS-independent functions (Min et al., 2010; White et al., 2012).
Together, this illustrates that RAS GAPs play diverse and discrete, yet non-redundant, roles in cell
signaling. The existence of distinct sets of modular domains in different RAS GAP proteins further
indicates that RAS GAP activity is highly complex and very tightly regulated. Nevertheless, still

Fig. 1. The RAS GTPase cycle and its deregulation in cancer. (A) RAS proteins together with their two key regulators, guanine
nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), constitute molecular switches that cycle between on and
off conformations caused by the binding of GTP or GDP, respectively. (B) Activating mutations in RAS genes occur in many different
tumor types. These mutations typically render RAS constitutively GTP-bound, resulting in the activation of downstream effector
pathways in the absence of extracellular stimuli. (C) Increasing evidence suggests that inactivation of several RAS GAP family
members represents an important and alternative mechanism to (hyper)activate RAS and its downstream effectors in cancer.
 O. Maertens, K. Cichowski / Advances in Biological Regulation 55 (2014) 114 3

Fig. 2. The RAS GAP family. There are 14 predicted RAS GAP genes in the human genome. All contain a RAS GAP domain but share
little similarity outside of this region. Members of the IQGAP subfamily contain an alternative amino acid within a key region of the
catalytic domain, and therefore do not exhibit RAS GAP activity but still affect RAS signaling.

relatively little is known about the precise function and regulation of many of these RAS GAP proteins
in normal processes or cancer. Moreover, the fundamental question of why so many RAS GAPs exist is
still not well understood.
The most extensively studied RAS GAP is neurobromin, the protein product of the NF1 gene
(Cichowski and Jacks, 2001). Despite the fact that germline mutations in NF1 were known to underlie
the cancer predisposition syndrome neurobromatosis type 1 (NF1), until recently a role for NF1 in
sporadic cancers had not been appreciated. Through genomic, cellular, and mouse modeling studies it
has now become evident that the NF1 gene/protein plays a much broader role in cancer than previously
recognized. There is also accumulating evidence that other RAS GAPs, such as DAB2IP, RASAL2, and
possibly RASA1, IQGAP2 and RASAL1 may function as bona de tumor suppressors and a surprising pro-
tumorigenic role has been proposed for IQGAP1. In this review, we will summarize studies describing
aberrant RAS GAP function in cancer for which sufcient validation supports a causal role in tumor
growth and metastasis. We will also highlight the emerging role of RAS GAPs in mediating resistance to
targeted therapies. Finally, we will address the mechanisms that alter RAS GAP gene or protein
expression in malignant transformation.

NF1: A tumor suppressor in familial and sporadic cancers

The NF1 gene and protein

The NF1 gene was identied by positional cloning in 1990 and encodes the RAS GAP known as
neurobromin (Ballester et al., 1990; Martin et al., 1990; Xu et al., 1990). Neurobromin is ubiquitously
expressed, although it is most abundant in the nervous system. Beyond the RAS GAP domain and the
immediately adjacent lipid-binding SEC14-PH domain (DAngelo et al., 2006; Welti et al., 2007), no
obvious protein motifs have been recognized in neurobromin. As the RAS GAP domain constitutes
only a small portion of the total protein (w13%), it has been suggested that other non-RAS associated
functions might exist. To this end, studies in Drosophila have shown that neurobromin can regulate
the cAMP pathway by regulating adenylyl cyclase activity (Guo et al., 2000; Tong et al., 2002; Walker
et al., 2013), although the molecular mechanism by which this occurs remains elusive.

Neurobromatosis type 1

Germline mutations and deletions of the NF1 gene underlie the familial cancer syndrome neuro-
bromatosis type 1 (NF1), which affects 1 in 3500 individuals worldwide (Riccardi, 1992). The hallmark
4 O. Maertens, K. Cichowski / Advances in Biological Regulation 55 (2014) 114

clinical feature is the development of peripheral nervous system tumors known as neurobromas.
While these lesions are typically benign, patients can develop hundreds of tumors throughout the
peripheral nervous system, which can be painful, debilitating and in some instances lethal. Moreover,
approximately 10% of individuals with NF1 will develop a malignant peripheral nerve sheath tumor
(MPNST). NF1 patients are also predisposed to developing a variety of other tumors, including gliomas,
pheochromocytomas and myeloid leukemia (Riccardi, 1992). Non-cancerous symptoms of the disease
include intellectual decits, bone deformations and hyperpigmentation defects such as caf-au-lait
spots and Lisch nodules. This broad spectrum of clinical manifestations in NF1 patients highlights the
requisite function of neurobromin in a wide variety of tissues and cell types.
In most instances NF1 behaves as a classical tumor suppressor gene. Genomic studies and mouse
models have shown that loss of heterozygosity (LOH) or second hit NF1 mutations are required for the
development of neurobromas, MPNSTs, pheochromocytomas and myeloid malignancies (Cichowski
et al., 1999; Jacks et al., 1994; Legius et al., 1993; Rutkowski et al., 2000; Serra et al., 2000; Shannon
et al., 1994; Vogel et al., 1999; Xu et al., 1992; Zhu et al., 2002). More than two decades of work has
now revealed critical insight relating to tumor cells of origin, cooperating genetic events, non-cell
autonomous signals, and molecular pathways underlying the pathophysiology of these NF1-related
tumors (Upadhyaya and Cooper, 2012). For example, loss of the wild-type NF1 allele in Schwann cell
precursors is the rate-limiting step for neurobroma development; however in some settings tumor
development requires NF1 haploinsufcency of surrounding cells within the microenvironment (Le
et al., 2011; Maertens et al., 2007; Mayes et al., 2011; Serra et al., 2000; Wu et al., 2008; Zheng et al.,
2008; Zhu et al., 2002). We also know that additional cooperating genetic lesions, such as P53 or
INK4A/Arf loss, are required for the formation of malignant gliomas and MPNSTs (Cichowski et al.,
1999; Joseph et al., 2008; Kourea et al., 1999; Legius et al., 1994; Nielsen et al., 1999; Vogel et al.,
1999; Zhu et al., 2005). In all instances it is clear that RAS-GTP levels are elevated in NF1-decient
lesions and that the downstream MAPK and PI3K/AKT pathways are generally activated as a conse-
quence of NF1 loss (Basu et al., 1992; DeClue et al., 1992; Johannessen et al., 2005; Lau et al., 2000).
Preclinical studies have further demonstrated the requirement for these RAS effector pathways in NF1-
mutant tumors (Chang et al., 2013; Dasgupta et al., 2005; De Raedt et al., 2011; Jessen et al., 2013;
Johannessen et al., 2005). Interestingly however, it appears that different effector pathways may be
dominant in different tumor types. For example while PI3K/mTOR appears to be the dominant pathway
in MPNSTs and gliomas (Dasgupta et al., 2005; Johannessen et al., 2005), the MAPK pathway is more
critical in benign neurobromas and JMML (Chang et al., 2013; Jessen et al., 2013).

NF1 loss in sporadic cancers

Recently, it has become evident that the NF1 gene/protein plays a much broader role in cancer than
previously recognized. High throughput genomic studies suggest that NF1 is mutated and/or inacti-
vated in a wide range of sporadic tumors, such as glioblastoma, lung adenocarcinoma, breast cancer,
leukemia, ovarian cancer and neuroblastoma (Boudry-Labis et al., 2013; Ding et al., 2008; Hodis et al.,
2012; Holzel et al., 2010; Krauthammer et al., 2012; McGillicuddy et al., 2009; Parsons et al., 2008;
TCGA, 2008; TCGA, 2011; TCGA, 2012). Although additional functional studies are required to under-
stand the biological signicance of many of these observations, cellular and animal models have
provided mechanistic insight into how NF1 cooperates with other genetic events in glioblastoma and
melanoma (Maertens et al., 2013; McGillicuddy et al., 2009; Zhu et al., 2005). Importantly, NF1 alter-
ations are not always mutually exclusive with RAS mutations or aberrations in RAS downstream
effector pathways such as BRAF or PTEN. Moreover, the mechanism of NF1 inactivation and its resulting
expression levels seem to be dictated by the genetic context (McGillicuddy et al., 2009).
In sporadic human gliomas, for example, NF1 has been shown to be inactivated via two distinct
mechanisms: proteasomal degradation and genetic loss (McGillicuddy et al., 2009). Notably, complete
genetic inactivation can only be tolerated in the presence of P53 mutations (McGillicuddy et al., 2009).
When P53 is intact, NF1-decient tumor cells become senescent in vitro and in vivo, as observed in
benign neurobromas and astrocytomas from NF1 patients (Riccardi, 1992). Loss of NF1 thus triggers a
senescence response, which restricts further progression of benign lesions (Courtois-Cox et al., 2006).
In order to evade senescence, additional genetic defects are required. This explains why bi-allelic
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inactivation of NF1 occurs in the context of P53 loss in NF1-related malignant gliomas and MPNSTs
(Cichowski et al., 1999; Vogel et al., 1999; Zhu et al., 2005), as well as in sporadic gliomas (McGillicuddy
et al., 2009). However, neurobromin can also be excessively degraded by the proteasome. Importantly,
neurobromin instability promotes tumorigenesis but is not sufcient to trigger a senescence response
(McGillicuddy et al., 2009). These ndings illustrate how different mechanisms of inactivation may be
required in genetically distinct tumors, in order to evade the senescence response.
Likewise, oncogene induced senescence is known to be important for restricting melanoma
development in response to activating BRAF mutations. Specically, oncogenic RAF can trigger a potent
negative feedback loop that suppresses RAS and downstream PI3K/AKT signaling, which plays a causal
role in the senescent response (Courtois-Cox et al., 2006; Maertens et al., 2013). Studies using genet-
ically engineered mouse models have demonstrated that in this setting Nf1 loss actually prevents Braf-
induced senescence of melanocytes, by relieving RAS and consequential PI3K repression (Maertens
et al., 2013). In mice, compound mutations in BRaf and Nf1 trigger excessive melanocyte hyper-
proliferation, which requires PI3K activation, and ultimately drive melanoma development. Impor-
tantly, NF1 mutations co-occur with BRAF mutations in human melanoma, and neurobromin
reconstitution potently suppresses the growth of human melanoma cells as xenografts, further sup-
porting a causal role for NF1 loss in melanomagenesis (Hodis et al., 2012; Krauthammer et al., 2012;
Maertens et al., 2013; Nissan et al., 2014). However, NF1 mutations also occur in the absence of acti-
vating BRAF mutations and can also co-occur with activating NRAS mutations (Hodis et al., 2012;
Krauthammer et al., 2012; Maertens et al., 2013; Nissan et al., 2014). This may be due to the nding
that, at least in melanoma, NF1 loss activates K- and H- but not N-RAS (Maertens et al., 2013). This
observation illustrates how NF1 mutations differ from activating mutations in a single RAS allele and
has important implications for mechanisms leading to drug resistance.

NF1 and resistance to targeted therapies

Targeted therapies directed against genetically well-dened vulnerabilities in human tumors have
been validated in the clinic. However, the relatively rapid acquisition of resistance to such treatments in
virtually all cases signicantly remains a substantial challenge to the clinical management of advanced
cancers (Lackner et al., 2012). As such, identifying mechanisms of resistance to inform the development
of second-line therapies or more durable rst-line treatments is of paramount importance.
Accumulating evidence suggests that NF1 plays an important role in mediating resistance to tar-
geted therapies. More specically, NF1 loss has been shown to drive resistance to kinase inhibitors in a
number of clinical settings, including BRAF-driven melanoma and EGFR-driven lung cancer (de Bruin
et al., 2014; Maertens et al., 2013; Nissan et al., 2014; Van Allen et al., 2014; Wang et al., 2014; Whittaker
et al., 2013). Genome-wide functional screens further identied loss of NF1 as a mediator of resistance
against tamoxifen in breast cancer cell lines and dasatinib in lung cancer cell lines (Beauchamp et al.,
2014; Mendes-Pereira et al., 2012). Elevated activation of RAS and its downstream pathways has been
shown to be the underlying mechanism of acquired resistance in all these cases. It is therefore tempting
to speculate that reduced expression levels of other functional RAS GAPs may also affect the sensitivity
to kinase inhibitors or different drugs in other cancers. Notably, while NF1 mutations have been
identied in resistant tumors and cell lines, loss of NF1 mRNA and protein expression appears to be
more common than bi-allelic alterations (de Bruin et al., 2014; Van Allen et al., 2014; Whittaker et al.,
2013).

Neurobromin inactivation by the proteasome

Despite its identication as a RAS GAP over 20 years ago, the regulation of neurobromin activity is
still not entirely understood. Neurobromin has been reported to be phosphorylated, but the func-
tional consequences of these phosphorylation events are unknown (Feng et al., 2004; Izawa et al., 1996;
Tokuo et al., 2001). Interestingly however, neurobromin is acutely degraded by the ubiquitin pro-
teasome pathway in response to a variety of growth factors through the activation of protein kinase C
(Cichowski et al., 2003; McGillicuddy et al., 2009). Importantly, these studies also demonstrated that
neurobromin degradation is essential for maximal RAS activation in response to many growth factors
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and that its re-expression is required to appropriately attenuate RAS signaling (Cichowski et al., 2003).
More recently the Cullin 3 and KBTBD7 (kelch repeat and BTB domain-containing 7) ubiquitin ligase
complex has been shown to control both the regulated proteasomal degradation of neurobromin and
the pathogenic destabilization of neurobromin in glioblastomas (Hollstein and Cichowski, 2013).
Together these ndings may have important clinical implications, as proteasome inhibitors, Cullin
inhibitors, PKC inhibitors and neddylation inhibitors would be expected to stabilize neurobromin and
therefore could be evaluated in tumor types such as glioblastoma, where neurobromin is inactivated/
degraded by the proteasome.

Additional mechanisms of neurobromin regulation

The mechanism by which neurobromin is recruited to specic intracellular compartments to

regulate growth factor induced RAS signaling also remains unclear; however, neurobromin contains a
lipid-binding SEC14-PH domain, which likely contributes to membrane localization (DAngelo et al.,
2006; Welti et al., 2007). Interestingly, recent work suggests that SPRED1 recruits neurobromin to
active signaling complexes at the plasma membrane (Stowe et al., 2012). Members of the SPRED
(Sprouty related protein with an N-terminal EVH1 domain) family turn off RAS signaling following
receptor kinase activation (Kim and Bar-Sagi, 2004). Specically, SPRED1 has been reported to recruit
neurobromin to the membrane via its EVH1 domain. From this location neurobromin promotes the
hydrolysis of RAS-GTP (Stowe et al., 2012). Notably, SPRED proteins also bind to c-KIT, and perhaps to
other receptor tyrosine kinases, suggesting that neurobromin regulates RAS locally in response to
specic receptor signaling, rather than simply suppressing RAS throughout the plasma membrane.
Notably, mutations in the SPRED1 gene underlie a syndrome that is clinically related to neurobro-
matosis type 1 (Brems et al., 2007). As such, the overlapping features between both clinical entities may
be explained by this direct interaction (Stowe et al., 2012).
Taken together, it has become clear that the NF1 tumor suppressor plays a broad and important role in
familial as well as sporadic cancer. Further work on the regulatory control and structural domain analysis
of neurobromin will have a profound impact on our mechanistic understanding of the precise function
of neurobromin in different tissues and contexts, and hence its biological consequences when lost.

DAB2IP: A tumor and metastasis suppressor that functions by concurrently regulating different
oncogenic pathways

DAB2IP loss promotes its effects via both RAS-dependent and -independent pathways

DAB2IP is a second important RAS GAP tumor suppressor to be validated as a human tumor sup-
pressor. However, while genetic alterations in DAB2IP do occur in human cancers, the DAB2IP gene
appears to be more frequently inactivated by epigenetic silencing and is suppressed in lung, breast,
prostate, hepatocellular and gastrointestinal tumors (Chen et al., 2005; Dote et al., 2005, 2004; Yano
et al., 2005; Zhang et al., 2012). Functional studies have now specically revealed a direct causal role
for DAB2IP loss in driving metastatic prostate cancer (Min et al., 2010; Wu et al., 2014; Xie et al., 2010).
However, DAB2IP appears to mediate its effects via both RAS-dependent and -independent pathways,
at least in this tumor type.
Activating RAS mutations are rare in prostate cancer, despite the fact that RAS effector pathways are
frequently activated and their activation correlates with progression and metastasis (Gioeli et al., 1999;
Jeong et al., 2008; Malik et al., 2002; Shen and Abate-Shen, 2007). Studies using an orthotopic model of
prostate cancer further demonstrated that HRASV12 could drive prostate cancer in the presence of
cooperating immortalizing genetic alterations (Berger et al., 2004). Because DAB2IP appeared to be
epigenetically silenced in prostate cancer cell lines, a functional role for DAB2IP inactivation in prostate
cancer was formally investigated. Interestingly, while HRASV12 promoted the development of high-
grade prostate adenocarcinomas in the orthotopic model described above, DAB2IP suppression simi-
larly drove tumor development but also triggered widespread metastasis (Min et al., 2010). These
results suggested that DAB2IP loss was more potent than an activated RAS allele and/or that additional
pathways were activated in response to DAB2IP suppression.
 O. Maertens, K. Cichowski / Advances in Biological Regulation 55 (2014) 114 7

As a member of the SynGAP family DAB2IP contains a PH domain and a single C2 domain both
N-terminal to the RAS GAP domain (Fig. 2). While both domains are thought to promote consti-
tutive plasma membrane association, a third more C-terminal period-like domain has been shown
to mediate an interaction with TRAF2, which suppresses NF-kB signaling (Zhang et al., 2004). As
such, loss of DAB2IP results in the activation of both the RAS and NF-kB pathways (Min et al., 2010).
Additional genetic and functional studies have now shown that in prostate cancer DAB2IPs effects
on RAS (through the RAS GAP domain) regulate primary tumor development, whereas its effects on
NF-kB (through the period-like domain) regulate metastasis (Min et al., 2010). Interestingly, DAB2IP
has also been reported to interact with GSK3b and PP2A via its C2 domain (Xie et al., 2010).
Therefore, while additional functional studies are required to fully elucidate DAB2IP function, this
RAS GAP appears to serve as a signaling scaffold that coordinately regulates two or three prominent
oncogenic pathways.

Mechanisms of DAB2IP inactivation in cancer

While DAB2IP mutations and deletions are found in different types of cancer (Barretina et al.,
2012; Forbes et al., 2011), epigenetic suppression seems to be the more prevalent mechanism of
inactivation. For example, DAB2IP is a target of the polycomb repressive complex 2 (PRC2)
(Schwartz and Pirrotta, 2013; Yu et al., 2007). Functional studies have further shown that EZH2,
which encodes the histone methyltransferase component of PRC2, is overexpressed in many cancer
types, including prostate cancer, and its expression correlates with tumor grade and metastasis
(Simon and Lange, 2008). Chromatin immunoprecipitation, functional studies and genomic analysis
have shown that EZH2 drives prostate tumorigenesis and metastasis, in part, via transcriptional
silencing of DAB2IP (Min et al., 2010). However, like many PRC2 targets, DAB2IP may ultimately
become more stably silenced by methylation of promoter sequences (Dote et al., 2005, 2004; Yano
et al., 2005). Thus the signicance of sequential silencing via histone and DNA modications should
be investigated further.

RASAL2: another tumor and metastasis suppressor

RAS mutations are also relatively uncommon in breast cancer, despite the fact that RAS effector
pathways are hyperactivated in the most advanced tumors (Forbes et al., 2011; Mueller et al., 2000;
Sivaraman et al., 1997; von Lintig et al., 2000). However, the RAS GAP gene RASAL2 has now been
shown to function as a tumor suppressor in breast cancer through its effects on RAS (McLaughlin et al.,
2013). Interestingly, Rasal2 loss also generally promotes the metastasis of tumors in p53 mutant mice,
suggesting that it mat play a broader role in tumor development and/or progression (McLaughlin et al.,
2013).
RASAL2 was initially identied as a potential RAS GAP tumor suppressor in a functional cell-based
screen (Min et al., 2010). Interestingly, mutations in the catalytic RAS GAP domain of RASAL2 were
observed in a small but signicant percentage of breast tumors, suggesting its putative role in this
setting (Sjoblom et al., 2006). Therefore a functional role for RASAL2 inactivation was formally inves-
tigated. By performing gain- and loss-of-function studies in human xenografts and genetically engi-
neered mouse models, McLaughlin et al. demonstrated that RASAL2 suppression promoted breast
tumor development as a consequence of activating K- and H-RAS. Moreover, in a luminal model of
breast cancer, RASAL2 mutations promoted metastasis (McLaughlin et al., 2013).
Like DAB2IP, RASAL2 appears to be more frequently silenced by epigenetic mechanisms in breast
cancer. Specically, its suppression (via promoter methylation) was shown to be enriched in luminal B
tumors (McLaughlin et al., 2013). Immunohistochemical and protein array analysis further demon-
strated that low RASAL2 protein levels are associated with metastasis (McLaughlin et al., 2013).
Moreover, low RASAL2 mRNA levels correlate with recurrence and poor overall survival of individuals
with luminal B cancers (McLaughlin et al., 2013). These observations may provide a mechanistic
explanation why luminal B tumors are more aggressive and refractory to therapies than luminal A
lesions. Additional studies evaluating the role of RASAL2 and other RAS GAPs in breast cancer will be an
important endeavor in the future.
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IQGAP1/2: scaffolding proteins with opposing effects

The IQGAP protein subfamily consists of three members with considerable sequence homology but
variable tissue distribution (Brill et al., 1996; Wang et al., 2007; Weissbach et al., 1994). The IQGAP
proteins are scaffolding proteins that contain multiple protein-interacting domains (White et al., 2012).
These regions include an actin-binding calponin homology domain (CH), a polyproline-binding domain
(WW), four IQ calmodulin-binding motifs (IQ) and a RAS GAP-related domain (Fig. 2). The RAS GAP-
related domain lacks an arginine residue essential for GTP hydrolysis and therefore none of the IQGAP
proteins exhibit RAS GAP activity (Brill et al., 1996; Wang et al., 2007; Weissbach et al., 1994). Interest-
ingly, this RAS GAP subfamily appears to be comprised of both oncogenes and tumor suppressors.
The best characterized member of the IQGAP family is IQGAP1. Through its interaction with specic
proteins IQGAP1 is implicated in multiple fundamental cellular activities (White et al., 2012). For
example, IQGAP1 binds to the Rho GTPases Rac1 and Cdc42 to regulate the assembly of the cyto-
skeleton (Erickson et al., 1997) and combines with microtubule-associated proteins to regulate cell
polarization and determine the direction of cell movement (Fukata et al., 2002). Furthermore, IQGAP1
binds members of the MAPK and PI3K/AKT signaling pathways to mediate cell proliferation and dif-
ferentiation (Bourguignon et al., 2005; Ren et al., 2007; Roy et al., 2004, 2005) and works with
calmodulin, b-catenin and E-cadherin to regulate intercellular adhesion and migration (Fukata et al.,
1999; Ho et al., 1999; Kuroda et al., 1998). Many of the identied IQGAP1 binding partners have
well-dened roles in tumorigenesis (White et al., 2009). This observation, together with the ability of
IQGAP1 to modulate a wide variety of cellular processes, suggests that IQGAP1 may be involved in
various aspects of tumor biology.
Accumulating evidence indeed suggests a positive role for IQGAP1 in cancer. Specically, IQGAP1 is
overexpressed and/or amplied in colorectal cancer, pancreatic adenocarcinoma, glioblastoma and
ovarian cancer, and its expression is correlated with poor prognosis (Dong et al., 2006; McDonald et al.,
2007; Nabeshima et al., 2002; Wang et al., 2013). Similar observations have been reported in mouse
models. Overexpression of Iqgap1 is associated with progression in a genetically engineered mouse
model that recapitulates the different stages of human prostate cancer (Ouyang et al., 2008). More
recently, IQGAP1 has been shown to function as a scaffold that is required for RAS-driven tumori-
genesis and metastasis. Specically, Iqgap1/ mice were resistant to Hras-driven chemical carcino-
genesis and depletion of IQGAP1 reduced the invasive properties of RAS-driven cancer cells (Jameson
et al., 2013). Nevertheless, the precise mechanism by which IQGAPs may directly contribute to
tumorigenesis and the mechanisms triggering IQGAP1s abnormal expression in cancer remain to be
elucidated. Notably, IQGAP1 appears to be dispensable for homeostasis and disruption of IQGAP1
binding to ERK diminishes tumorigenesis in vivo (Jameson et al., 2013), suggesting a therapeutic
window could be achieved by targeting IQGAP1 in some cancers.
In contrast to IQGAP1s apparent pro-tumorigenic role, IQGAP2 was found to be inactivated by pro-
moter methylation in gastric cancer (Jin et al., 2008) and Iqgap2 deciency leads to the development of
hepatocellular carcinoma in mice (Schmidt et al., 2008). Remarkably, concurrent Iqgap1 loss partially
rescues the tumor phenotype of Iqgap2/ mice and a reciprocal relationship exists between IQGAP1 and
IQGAP2 expression in human liver cancer cell lines (Schmidt et al., 2008; White et al., 2010). Together,
these data suggest that IQGAP1 and IQGAP2 have opposing oncogenic and tumor suppressive roles.
While there is no evidence for a direct interaction between both proteins, their relative expression and/
or stoichiometry might determine various cellular and biological consequences. Further studies are
needed to elucidate the relative contribution, specic binding partners, subcellular localization, distinct
tissue expression and post-translational modications of IQGAP1 and IQGAP2. This will help provide
insight into the seemingly paradoxical roles of these two very similar IQGAP proteins in cancer. Similarly,
additional work is necessary to determine the possible role of IQGAP3 in neoplasia.

Other RAS GAPs in cell signaling and cancer

P120 RAS GAP was the rst protein to be characterized as a RAS GAP (Trahey et al., 1988; Vogel et al.,
1988). In addition to the RAS GAP domain P120 contains SH2, SH3, PH, and C2 domains (Fig. 2). All four
domains are thought to be involved in targeting P120 to membranes, and initial studies suggested that
 O. Maertens, K. Cichowski / Advances in Biological Regulation 55 (2014) 114 9

P120 can associate with receptor tyrosine kinases. For example, SH2 domains enable binding of P120 to
phosphorylated epidermal growth factor receptor, platelet derived growth factor receptor, and insulin
receptors which promotes inactivation of RAS in the respective signaling pathways (Agazie and
Hayman, 2003; Cooper and Kashishian, 1993; Ekman et al., 1999; Fantl et al., 1992; Kazlauskas et al.,
1990; Margolis et al., 1990). There is also evidence that the SH3 domain of P120 interacts with DLC1
and as such interferes with the Rho GAP activity of this protein (Yang et al., 2009). Although mutations
in the RASA1 gene, which encodes P120, have been found in individuals affected with a capillary and
arteriovenous malformation syndrome (Eerola et al., 2003), its link with cancer has remained more
obscure. An early genetic study reported somatic RASA1 mutations in basal cell carcinoma (Friedman
et al., 1993) and integrative bioinformatic analysis identied RASA1 as a candidate tumor suppressor
gene associated with triple negative and HER2 positive subtypes of breast cancer (Hu et al., 2009). A
recent computational study analyzing the patterns of mutational signatures in a broad panel of tumors
also predicts RASA1 to be a putative tumor suppressor (Davoli et al., 2013).
Somatic inactivation of RASAL1 by epigenetic silencing and mutations has been reported in a
number of human malignancies, including colorectal, thyroid and gastric cancer and nasopharyngeal
and esophageal tumors (Jin et al., 2007; Liu et al., 2013; Ohta et al., 2009; Seto et al., 2011). Knockdown
of RASAL1 as well as expression of RASAL1 mutants in cell lines resulted in RAS activation, increased
signaling via downstream MAPK and PI3K/AKT effectors, and accelerated growth of xenografted tu-
mors. Conversely, ectopic expression of wild-type RASAL1 had the opposite effects (Liu et al., 2013;
Ohta et al., 2009; Seto et al., 2011). Moreover, a genetic screen identied the transcription factor
PITX1 as a suppressor of RAS activity and tumorigenicity by upregulating RASAL1 (Kolfschoten et al.,
2005). Together, these data indicate that RASAL1 might function as a bona de tumor suppressor by
regulating RAS activity and that PITX1 may be one of the factors mediating its aberrant expression in
cancer. Reduced RASAL1 expression is detected more frequently in advanced lesions, suggesting that
RASAL1 might be involved in both tumor initiation and progression (Liu et al., 2013; Ohta et al., 2009;
Seto et al., 2011). Additional studies are required to formally demonstrate a causative role for RASAL1
loss in cancer.

Inactivation mechanisms of RAS GAPs in cancer: caveats and opportunities

Unlike the frequent direct mutational activation of RAS, a preponderance of evidence suggests that
RAS GAPs are commonly deregulated by non-genetic mechanisms. These non-mutational inactivation
mechanisms include epigenetic silencing, transcriptional repression and proteasome-mediated
degradation. Additional mechanisms are likely to be discovered. Regulation of RAS GAP expression
by non-coding RNAs, for example, remains an unexplored research area. Furthermore, post-
translational modications, proteinprotein interactions and aberrant localization can be expected
to positively or negatively regulate RAS GAP function. It is very likely that, as has been shown for PTEN,
the regulation of RAS GAPs by various molecular mechanisms allows to generate a continuum of
functional RAS GAP levels with differential consequences in inherited syndromes and sporadic cancers
(Song et al., 2012).
As such, protein analysis, rather than mutational or copy number status, might represent a more
accurate way to assess the true frequency of RAS GAP inactivation in cancer. The development of
reliable antibodies against specic members of the RAS GAP family, and guidelines on how to use these
in a clinical setting, is therefore of high priority. It will allow to evaluate the RAS GAP status in clinical
samples. Given the association of particular RAS GAPs with metastasis, poor prognosis and resistance
to targeted therapies, this assay could be used as a useful biomarker during clinical trials and for
routine pathological staining.
In contrast to the pro-tumorigenic IQGAP1 gene, the remaining RAS GAP tumor suppressor genes are
not considered classically druggable targets because it is traditionally easier to develop small molecule
antagonists rather than agonists. However, the observation that RAS GAPs are predominantly inacti-
vated by non-mutational mechanisms represents a major therapeutic opportunity. In principle,
restoring the expression of the wild-type (and functional) RAS GAP could be clinically exploited as a
strategy to inhibit the activation of wild-type RAS in particular cancers. For example, the insight that
neurobromin is degraded by the ubiquitin-proteasome pathway in glioblastoma suggests that small
10 O. Maertens, K. Cichowski / Advances in Biological Regulation 55 (2014) 114

molecule inhibitors aimed at blocking its destruction, would be expected to work in these tumors
(Hollstein and Cichowski, 2013; McGillicuddy et al., 2009). Similar approaches targeting the protea-
somal degradation of P53 by small molecules such as nutlin-3, which inhibits p53-MDM2 binding, have
been an active area of therapeutic development in hematologic malignancies and neuroblastoma
(Cheok et al., 2011).
Gaining further critical insight into the different mechanisms that alter RAS GAP expression in
cancer is therefore of particular importance. It should not only reveal prognostic biomarkers but can
also point towards the most druggable targets for therapeutic intervention. In the meantime the
challenge will be to develop a reliable means of quantifying RAS GAP suppression, and to better un-
derstand the threshold and consequences of reduced RAS GAP levels, so that these insights may ul-
timately be exploited in the clinic.

Concluding remarks

A growing body of evidence suggests that several RAS GAP proteins play an important and previ-
ously unrecognized role in cancer. In addition to genetic defects, it is clear that pathological mecha-
nisms that modulate RAS GAP gene or protein expression are associated with malignant
transformation. While ablation of RAS GAP activity represents an alternative mechanism to activate
RAS, the implications of RAS GAP loss appear to be more distinct and enigmatic and in some instances
promote the activation of additional oncogenic pathways. Further understanding of how the levels of
RAS GAP expression and activity are regulated, and what the biological consequences of RAS GAP
misregulation are, is therefore urgently needed. It can be expected that insights from these studies will
help shed light on the complex cellular signaling circuitry in normal biology and cancer.

Conicts of interest

The authors declare that there are no conicts of interest.

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