Download Tyrosine kinase receptor-activated signal transduction

Oncogene (1998) 16, 1343 ± 1352
 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00
http://www.stockton-press.co.uk/onc
Tyrosine kinase receptor-activated signal transduction pathways which lead
to oncogenesis
Amy C Porter and Richard R Vaillancourt
Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, USA
Oncogenesis is a complicated process involving signal
transduction pathways that mediate many di€erent
physiological events. Typically, oncogenes cause unregulated cell growth and this phenotype has been attributed
to the growth-stimulating activity of oncogenes such as
ras and src. In recent years, much research e€ort has
focused on proteins that function downstream of Ras,
leading to the identi®cation of the Ras/Raf/MAPK
pathway, because activation of this pathway leads to
cellular proliferation. Activated receptor tyrosine kinases
(RTKs) also utilize this pathway to mediate their growthstimulating e€ects. However, RTKs activate many other
signaling proteins that are not involved in the cellular
proliferation process, per se and we are learning that
these pathways also contribute to the oncogenic process.
In fact, RTKs and many of the proteins involved in
RTK-dependent signal transduction can also function as
oncogenes. For example, the catalytic subunit of
phosphoinositide 3-kinase (PI3-K) was recently identi®ed
as an oncogenic protein. The scope of pathways that are
activated by oncogenic RTKs is expanding. Thus, not
only do RTKs activate Ras-dependent pathways that
drive proliferation, RTKs activate PI3-K-dependent
pathways which also contribute to the oncogenic
mechanism. PI3-K can initiate changes in gene transcription, cytoskeletal changes through b-catenin,
changes in cell motility through the tumor suppressor,
adenomatous polyposis coli (APC), and phosphorylation
of BAD, a protein involved in apoptotic and antiapoptotic signaling. There is also cross-talk between
RTKs and the oncostatin cytokine receptor which may
positively and negatively in¯uence oncogenesis. For this
review, we will focus on oncogenic RTKs and the
network of cellular proteins that are activated by RTKs
because multiple, divergent pathways are responsible for
oncogenesis.
Keywords: phosphoinositide 3-kinase; cytoskeleton;
apoptosis
Introduction
Through the evolutionary process, retroviruses have
targeted genes whose products are essential for a cell's
normal physiological role within an organism. The
targeted genes, referred to as proto-oncogenes, encode
proteins that are key regulators for cell growth,
di€erentiation, survival or motility. Mutations in
Correspondence: RR Vaillancourt
proto-oncogenes disrupt the regulated activity of the
normal or wild-type gene product and the activity of
the mutated protein confers a selective advantage for
infected cells to survive over their non-infected
counterparts. As we identify and characterize more
proto-oncogenes and the proteins they encode, we are
gaining a better understanding of the mechanisms that
the wild-type proteins utilize to govern normal cellular
physiology and the mechanisms that the oncogenic
proteins utilize to ensure their evolutionary success.
However, it is important to remember that oncoproteins do not function alone in the cell. In other
words, the cellular signaling pathways that are
activated or inactivated by an onco-protein are equally
as important, as the onco-protein itself, for the
oncogenic process to occur.
Historically, the morphological transformation of
the mouse ®broblast cell line NIH3T3 has been used as
a screen for oncogenes. DNA derived from human
lung and colon tumors was transfected into NIH3T3
cells which led to the identi®cation of ras as a human
oncogene (Cooper, 1982; Weinberg, 1981). Further
characterization of the ras oncogene determined that
point mutations resulted in the activation of Ras, even
though the signal transduction pathway that involved
Ras had not been determined at the time (Reddy et al.,
1982; Tabin et al., 1982; Yuasa et al., 1983). It was
over a decade later that Ras was identi®ed as an
activator of Raf (Vojtek et al., 1993). These earlier
studies demonstrated that oncogenes are the mutated
forms of cellular proteins. Even today, a common
experimental approach to identify oncogenic genes is to
isolate DNA from tumor cells and compare the
nucleotide sequence relative to that of the protooncogene. This approach has led to the identi®cation
of receptor tyrosine kinases (RTKs) as proto-oncogenes.
In order for an oncogene to be tumorigenic, there
are many events that must occur within the cell. A
change in the proto-oncogene nucleotide sequence
occurs and the resulting gene product is a protein
whose activity is not regulated like its wild-type
counterpart. Cell proliferation has commonly been
associated with the unregulated activity of oncogenes
since cellular transformation is the phenotype that is
observed in tumors. However, as we have learned more
about signal transduction pathways and oncogenes, it
is fair to state that cellular proliferation may be
necessary, but it is not sucient, for a transformed
cellular phenotype. Due to the pleiotropic nature of
RTKs and the signaling proteins they activate, we are
®nding that maintenance of cell survival and motogenic
pathways also play an important role in oncogenic
signaling.
Oncogenic pathways
AC Porter and RR Vaillancourt
1344
In the past, Ras has received much attention due
to the high frequency of mutations that occur in the
ras gene that is isolated from a variety of tumors.
Because of this observation, scientists focused on
Ras-dependent pathways and found that many
RTKs produce a mitogenic signal through Ras.
However, it has become apparent that tumors have
properties that make them unique and that these
properties are mediated by Ras-independent pathways. As an example, RTKs activate PI3-K whose
lipid products function as second messengers to
activate Akt which phosphorylates BAD, producing
an anti-apoptotic signal for the cell. As a
consequence, RTK activation of Ras-dependent and
PI3-K-dependent pathways may contribute equally to
produce a proliferative and an anti-apoptotic
response, respectively. As a result of activating
these two divergent pathways, mutated cells are
allowed to selectively proliferate and those cells are
not removed by normal apoptotic mechanisms.
Therefore, multiple signaling pathways may be
necessary for cellular transformation and no one
pathway, alone, may be sucient to produce an
oncogenic response. In the future, our knowledge of
how these pathways are integrated may increase our
chances of developing successful therapeutic strategies to treat malignant cancers, and possibly, allow
us to develop strategies to prevent them from
occurring. This review will focus on the mechanisms
and pathways that cause RTKs and their substrate
proteins to function as oncogenic proteins.
Receptor tyrosine kinases
Clearly, receptor tyrosine kinases can function as
oncogenes. Over the past few years, numerous
excellent reviews have appeared on RTKs and the
`classical' SH2 (Src Homology 2) domain containing
pathways they activate (Cohen et al., 1995; Ullrich and
Schlessinger, 1990; van der Geer et al., 1994). More
recently, RTKs have been shown to utilize `novel'
mechanisms to generate signals (reviewed in Weiss et
al., 1997). As we learn more about RTK-dependent
signaling pathways, only time will allow us to
di€erentiate between classical and novel signaling
pathways for RTKs. For purposes of this review, we
will focus on RTKs and the signaling pathways they
utilize that allows them to function as oncogenes. Since
there are many oncogenic RTKs, we will discuss a
select few in this review to highlight oncogenic
mechanisms that involve RTKs.
RTKs are composed of an extracellular ligand
binding domain, a transmembrane domain, an
intracellular tyrosine kinase domain, and additional
amino acid sequences that function as regulatory
domains. Ligand binding induces receptor dimerization and autophosphorylation in a trans fashion, that
is, one subunit of the dimer phosphorylates the
opposing subunit. Phosphorylated tyrosines function
to recruit intracellular signaling proteins, many via
their SH2 domains. In addition, SH2-containing
proteins, such as Grb2, function as adaptor proteins
to recruit multiple proteins to RTKs, thus increasing
the repertoire of signaling proteins that are activated
by RTKs.
The Ras/Raf/MAPK pathway has received much
attention over the last few years because it plays a
major role in cellular proliferation. The link between
RTKs and Ras that Grb2 provides clearly demonstrates the importance of this pathway in RTK
signaling. However, we are learning more about the
network of signaling molecules that are activated by
RTKs. As a consequence, the physiological events that
these pathways regulate also contribute to the
oncogenic process. Accordingly, we need to characterize these pathways to gain a better understanding of
the mechanisms that oncogenic RTKs utilize
(Figure 1).
RET
Ret is a proto-type RTK oncogene because numerous,
di€erent mutations and gene rearrangements of the ret
proto-oncogene have been isolated from tumor cells.
For this reason, we will discuss Ret to illustrate the
types of mutations that can occur in RTKs. The c-ret
proto-oncogene encodes an RTK that also contains an
extracellular cadherin-like domain and an interrupted
tyrosine kinase domain, in addition to the aforementioned features of RTKs. The unglycosylated receptor
is 120 kDa while neuroblastoma cells express a 150 and
170 kDa form of the receptor (Takahashi et al., 1991,
1997). Ret expression occurs predominantly in neural
crest-derived cells. The biological function of Ret is not
well de®ned although expression studies suggest that
Ret is involved in cell migration, proliferation, and
di€erentiation (Pachnis et al., 1993; Tsuzuki et al.,
1995). Ret knock-out mice die early during neonatal
development and show severe renal abnormalities,
demonstrating the importance of the Ret protein for
embryonic development (Schuchardt et al., 1994).
Missense, nonsense, and gene rearrangement of the
ret proto-oncogene are the types of mutations that we
will discuss. When missense mutations occur, an amino
acid substitution is introduced into the resulting gene
product which causes the protein to function in an
unregulated manner, typically producing constitutive
activity. In contrast, nonsense mutations introduce a
stop codon within the coding sequence of the gene
causing incomplete translation of the protein, resulting
in a loss-of-function mutation. A gene rearrangement
occurs when pieces of DNA, typically from di€erent
chromosomes, fuse to produce hybrid genes and the
resulting protein activity is not regulated in a manner
that is similar to that of the wild-type proteins.
Although frameshift mutations occur in the ret protooncogene, they will not be discussed in this review.
Mutations in the c-ret proto-oncogene result in two
classes of genetic diseases (reviewed in Edery et al.,
1997; Mak and Ponder, 1996). The developmental
disorder, Hirschsprung's disease, is attributed to lossof-function mutations, while activating point mutations
are associated with autosomally dominant inherited
cancer syndromes of three types, multiple endocrine
neoplasia (MEN) type 2A, MEN type 2B, and familial
medullary thyroid carcinoma (FMTC). The extracellular domain of Ret is a hot spot for mutations.
Individuals with the MEN 2A and FMTC syndromes
have mutations in a cluster of ®ve cysteine codons in
the extracellular domain of ret, (C609?R/W/Y),
Oncogenic pathways
AC Porter and RR Vaillancourt
(C611?Y/W), (C618?G/F/S/R/Y), (C620?G/F/S/R/
Y), (C634?G/F/S/R/Y/W) (Donis-Keller et al., 1993;
Mulligan et al., 1993). The net result of each of these
missense mutations is ligand-independent activation of
Ret tyrosine kinase activity. Additional missense
mutations occur at codons 768 and 804 which are
localized to the kinase domain and the kinase insert
domain of the receptor. These mutations have been
identi®ed from a few FMTC families demonstrating an
oncogenic consequence of these mutations (Bolino et
al., 1995; Eng et al., 1995). However, the signaling
pathways that are activated by these mutant receptors
have not been characterized. The MEN 2B syndrome,
although less common than MEN 2A, is associated
with a M?T mutation at codon 918 of the second part
of the tyrosine kinase domain (Carlson et al., 1994;
Hofstra et al., 1994). Similarly, the signaling pathway
that mediates the oncogenic response for this receptor
is not known, although constitutive activation of a
signaling pathway probably mediates the oncogenic
activity of this receptor.
In addition to speci®c point mutations, the ret
proto-oncogene undergoes gene rearrangement. This
phenomena was observed when DNA from human T
cell lymphoma was transfected into NIH3T3 cells.
DNA from the 3' end of the ret gene fused to the 5'
end of unrelated DNA sequence, hence the name RET
REarranged during Transfection, (Takahashi et al.,
1985). Although these DNA rearrangements were due
to the experimental method, this ®nding suggested that
the ret gene has a tendency to undergo DNA
rearrangement. In fact, rearrangement of the 3'
sequence of the ret proto-oncogene with the 5'
sequences of H4/D10S170 (Bongarzone et al., 1996;
Grieco et al., 1990), the type Ia regulatory subunit of
cyclic AMP-dependent protein kinase (Bongarzone et
al., 1993), and the ELE1 gene (Santoro et al., 1994a)
have been detected from DNA extracts from papillary
thyroid carcinomas (PTC). The resulting hybrid genes
(Ret/ptc1, Ret/ptc2, and Ret/ptc3) consist of the Ret
tyrosine kinase domain fused with genes that are
capable of intracellular dimerization, thus producing
Figure 1 Multiple pathways contribute to cellular transformation. Oncogenic receptor tyrosine kinases are pleiotropic, activating numerous
intracellular signaling pathways. Grb2 and Shc (not shown) associate with activated receptors and participate in the activation of Ras, a
small molecular weight GTP binding protein that functions upstream of sequential protein kinases of the MAPK pathway. Activation of
MAPK is necessary for cellular proliferation, di€erentiation, and cell scattering. In addition to activation of MAPK, oncogenic Ras
stimulates the release of growth factors such as IGF-1. Acting in an autocrine fashion, IGF-1 stimulates its tyrosine kinase receptor which
recruits and activates PI3-K, via the insulin receptor substrate 1 (IRS-1), to produce the phospholipids PI(3,4,5)P3 and PI(3,4)P2. When
bound to the PH domain of Akt, these phospholipids activate Akt to phosphorylate BAD and GSK3. When phosphorylated, BAD is an
anti-apoptotic signal in cells. Akt phosphorylation of GSK3 results in inactivation of GSK3. This prevents the association of GSK3, APC
and free b-catenin which would normally result in degradation of b-catenin. Instead, b-catenin can go on to a€ect gene transcription leading
to proliferation and/or activation of anti-apoptotic pathways and APC can go on to play a role in cell motility. Oncogenic RTKs can also
phosphorylate b-catenin, involved in cell-cell adhesion and the cytoskeleton, resulting in loss of the interaction of b-catenin with the actin
cytoskeleton which could contribute to cell scattering. Thus, oncogenic RTKs are capable of using multiple, divergent mechanisms to
undermine the normal physiological functions of cells
1345
Oncogenic pathways
AC Porter and RR Vaillancourt
1346
hybrid proteins with a constitutively active Ret kinase.
The Ret/ptc1 gene product, unlike the wild-type
receptor, is localized to the cytoplasm and constitutively phosphorylated on tyrosine (Ishizaka et al.,
1992).
Only recently has the Ret ligand been identi®ed as
glial cell-derived neurotrophic factor (GDNF (Durbec
et al., 1996; Trupp et al., 1996)). Prior to this
discovery, a chimera of the extracellular ligand
binding domain of the EGF-R and the intracellular
domain of Ret was used to study Ret signaling
pathways. Stimulation of cells expressing the EGF-R/
Ret chimera resulted in phosphorylation of paxillin, a
cytoskeletal protein, and cellular transformation
(Romano et al., 1994; Santoro et al., 1994b). In
addition, stimulation of the EGF-R/Ret chimera in
SK-N-MC neuroepithelioma cells leads to cell scattering, growth inhibition, and loss of anchorageindependent growth (van Puijenbroek et al., 1997).
Characterization of the mitogen-activated protein
kinase (MAPK) pathway from these cells indicated
that ERK2 (extracellular signal-regulated kinase 2) was
activated in a sustained fashion, suggesting that cell
scattering correlates with sustained ERK2 activation.
In contrast, a mitogenic response from EGF in PC12
cells correlates with transient ERK activation (Heasley
and Johnson, 1992).
Studies with the Ret/ptc2 oncogene have identi®ed
phospholipase Cg (PLCg), Grb10 and Shc as signaling
proteins with SH2 domains that associate with
phosphorylated tyrosines of Ret/ptc2. Phosphotyrosine (pTyr)539, which is Tyr761 on the Ret protooncogene, and pTyr429 are the docking sites for PLCg
and Grb10, respectively (Borrello et al., 1996), while
pTyr586 associates with Shc (Arighi et al., 1997).
Although the biological role of Ret is not well de®ned,
the interaction of PLCg and Grb10 is not required for
mitogenic signaling of Ret/ptc2.
Enigma is unique in that it associates with Ret/ptc2
via unphosphorylated Tyr586 demonstrating that
unphosphorylated RTKs are capable of recruiting
signaling proteins (Durick et al., 1996). Additional
data to support this observation came from experiments where kinase-inactive Ret/ptc2 was found to
interact with Enigma in the yeast two-hybrid system,
demonstrating that the kinase activity of Ret/ptc2 is
not necessary for this interaction. However, the kinase
activity of Ret/ptc2, as well as an interaction between
Ret/ptc2 and Enigma, is necessary for Ret/ptc2
mitogenic signaling. The signaling pathways that are
regulated by Ret are beginning to emerge. Now that
many of the mutations in the ret proto-oncogene have
been identi®ed, a careful analysis of the signaling
pathways that are activated by the mutant proteins
may shed light on new oncogenic pathways or
reemphasize the importance of oncogenic pathways
that have already been de®ned.
Hepatocyte growth factor receptor
Invasive cell growth is a component of the oncogenic
process that contributes to the malignancy of tumor
cells. Stimulation of the hepatocyte growth factor
receptor (HGF-R) is particularly relevant to oncogenesis because this receptor stimulates invasive growth of
carcinoma cells (Weidner et al., 1990), is a potent
angiogenic agent (Bussolino et al., 1992), and is
tumorigenic (Rong et al., 1992). HGF, the ligand for
the HGF-R, is also known as scatter factor because it
stimulates cellular motility and functions as a
morphoregulatory agent. HGF is a heterodimer
composed of a 69 and a 34 kDa subunit which binds
to the 190 kDa HGF/scatter factor (SF) receptor.
HGF-R is a pleiotropic, epithelial cell receptor, capable
of stimulating mitogenesis, motility, invasive growth
and inducing branching tubulogenesis.
Following stimulation with HGF, many proteins
associate with the receptor, become phosphorylated
and/or become activated by the HGF-R. These
proteins include PI3-K (Royal and Park, 1995), PLCg
(Zhu et al., 1994), Grb2 (Zhu et al., 1994), Ras
(Graziani et al., 1993), focal adhesion kinase
(Matsumoto et al., 1994), b-catenin, plakoglobin
(Shibamoto et al., 1994) and Shc (Pelicci et al., 1995).
Many of these proteins are activated by mitogenic
growth factor receptors, such as the PDGF-R (Valius
and Kazlauskas, 1993). Like many RTKs, tyrosine
phosphorylation within the kinase domain is required
for catalytic activity (Longati et al., 1994).
Little is known about the signaling pathways
involving cell dissociation, motility, and morphogenesis that are mediated by the HGF-R. The HGF-R
recruits a number of e€ector proteins including Grb2,
Shc, the p85 regulatory subunit of PI3-K, PLCg and
the phosphatase SHP2 by association with pTyr1356
(Zhu et al., 1994). Grb2, Shc and PI3-K associate with
pTyr489 (equivalent to Tyr1356 of Met or HGF-R) of
the Tpr-Met oncoprotein (Fixman et al., 1995, 1996).
Functionally, Tyr1356 is also necessary for transformation of ®broblasts mediated by Tpr-Met. Given our
current understanding of the speci®city of SH2
domains, it seems counter intuitive to have so many
proteins bind to the same phosphotyrosine.
More recently, Nguyen et al. (1997) have shown that
phosphorylation of Tyr1356 is necessary for recruitment and phosphorylation of Gab1. However, association of Gab1 with the HGF-R is not direct, but
mediated primarily by Grb2, where Grb2 functions as
an adaptor between the two proteins. Thus, Gab1 may
function as a docking site for the p85 subunit of PI3-K,
PLCg, and SHP2 through Tyr1356. Of these signaling
proteins, PI3-K activity is essential for HGF-Rmediated cell dissociation and subsequent scattering
(Royal and Park, 1995).
There is con¯icting data regarding the role of
Tyr1356 in HGF-R signaling. Madin-Darby canine
kidney (MDCK) cells expressing the Y1356F mutant
HGF-R retain their ability to scatter but fail to
stimulate tubulogenesis of tubular epithelial cells
(Nguyen et al., 1997). In contrast, MDCK cells
expressing a receptor chimera composed of the CSF1R (extracellular domain)/Met (transmembrane and
intracellular domain) and treated with CSF-1 fail to
scatter, undergo invasive cell growth, and form
branching tubules (Pasdar et al., 1997). In addition,
stimulation of the CSF-Met receptor inhibits cell-cell
contact and junction formation, possibly by regulating
E-cadherin and plakoglobin (reviewed in Daniel and
Reynolds, 1997). E-cadherin may play a role in
invasive cell growth mediated by the HGF-R since it
is frequently down-regulated in metastatic tumors.
Oncogenic pathways
AC Porter and RR Vaillancourt
Receptor over-expression
One of the hallmarks of tumor cells is the overexpression of growth factor receptors. The epidermal
growth factor receptor (EGF-R) is not mutated nor
does it undergo gene rearrangement. However, overexpression of the EGF-R in many tumors, as well as
association with the erbB-2 (Her-2/neu) oncogene and
the c-cbl proto-oncogene, likely contributes to the
oncogenic potential of the EGF-R as a stimulator of
cell proliferation. In tumors, the EGF-R is frequently
over-expressed which produces an autocrine/paracrine
loop such that the mitogenic potential of EGF is
selectively enhanced in tumors as compared to normal
cells. Similarly, the HGF-R or the Met protein is overexpressed in tumors.
When comparing normal epithelial cells to epithelial
tumor cells, the HGF-R is over-expressed, up to 100fold in thyroid papillary carcinomas (Di Renzo et al.,
1992; Prat et al., 1991). To investigate a possible
mechanism to explain this observation, Ivan et al.
(1997) expressed activated Ras under the control of an
inducible promoter in the R18 human thyroid cell line
and found that HGF-R expression increased fourfold.
In a paracrine fashion, the role of increased HGF-R
expression would be to increase tumor sensitivity to
HGF secreted from adjacent stromal cells. However,
the authors found that this increased sensitivity to
HGF was not translated into a mitogenic response, as
one would predict. In fact, the role of increased HGFR expression may be to provide a motogenic signal to
tumor cells, allowing for invasive cell growth (reviewed
in Jiang & Hiscox, 1997).
Receptor dimerization
In order for a proto-oncoprotein to function as an
oncogene, a mutation must occur which changes the
regulated activity of the gene product. For RTKs,
localization to the plasma membrane may serve two
functions with respect to regulation of receptor
activity. One function would be to allow the receptor
to bind its respective growth factor, since the ligands
do not di€use across the plasma membrane. A second
function for localizing a RTK to the plasma membrane
may be to prevent or minimize unregulated activation
of cytoplasmic proteins due to the intrinsic, basal
tyrosine kinase activity of RTKs. Through mutations
or the process of gene rearrangement, oncogenic RTKs
bypass both of these functions.
The nerve growth factor receptor (NGF-R) undergoes a gene rearrangement with tropomyosin (MartinZanca et al., 1986), hence the name trk (tropomyosin
receptor kinase). The extracellular domain of the
NGF-R is lost and tropomyosin is fused to the kinase
domain. Similarly, the HGF-R undergoes gene
rearrangement when sequences from chromosome 1
called tpr (translocated promoter region) fuse upstream
with the HGF-R kinase domain (Cooper et al., 1984;
Park et al., 1986). The resulting Tpr-Met hybrid
protein lacks the extracellular and transmembrane
domain of the HGF-R. It was determined from sitedirected mutagenesis studies that the leucine zipper
motif of Tpr was necessary for dimerization of TprMet (Rodrigues and Park, 1993). The inability to form
dimers also correlated with the loss of autophosphorylation of Tpr-Met and focus forming activity.
Over-expression of p185erbB2 occurs in approximately 33% of primary breast and ovarian tumors. A
point mutation (V664?E) in the putative transmembrane domain renders p185erbB2 constitutively active,
leading to Shc phosphorylation and activation of
MAPK (Nowak et al., 1997). Analysis of the physical
properties of this mutant receptor determined that it is
capable of forming dimers. Also, a chimeric p185erbB2
receptor containing the transmembrane domain of
glycophorin A was capable of dimerization, suggesting
that amino acid sequences outside the transmembrane
domain are necessary for receptor dimerization (Burke
et al., 1997). Although the p185erbB2/glycophorin A
chimera could form a dimer, it was unable to induce a
transformed phenotype in cells. Thus, dimerization
may be necessary, but is not sucient for transforming
activity. These results suggest that constitutive or
unregulated kinase activity may be the driving force
that determines the oncogenic potential of RTKs and
that dimerization may be an important feature of
regulated RTK activity.
Oncostatin M
There is some indication that there is cross-talk
between the RTKs and the cytokine oncostatin M
receptor (OSM-R) which may play a role in
oncogenesis. Oncostatin M is a 28 kDa glycoprotein
cytokine produced by activated T cells, macrophages
and phorbol ester-treated U937 leukemia cells (Zarling
et al., 1986). OSM appears to act through a two
subunit cell surface receptor, typical of cytokine
receptors. The gp 130-signal transducing protein is a
shared component of the Type I and Type II OSM-R.
The Type I receptor consists of gp 130 and the
leukemia inhibitory factor receptor b (LIF-Rb) and
also binds leukemia inhibitory factor (LIF). The Type
II receptor consists of gp 130 and the 180 kDa
oncostatin M receptor b (OSM-Rb; gp 180) and does
not bind LIF. It is the Type II receptor which appears
to transduce most of the speci®c signals activated only
by OSM and not by LIF (Thoma et al., 1994). The two
subunits of the receptor form a dimer upon ligand
binding and are coupled to tyrosine phosphorylation,
although the receptor does not possess any endogenous
kinase activity (Ihle and Kerr, 1995). One of the many
e€ects of OSM is its ability to suppress growth of some
types of tumor cells (Liu et al., 1997a). However, OSM
is also able to stimulate growth of some types of
cancer, such as Kaposi's sarcoma (Miles et al., 1992;
Nair et al., 1992), while having no e€ect on other
types, such as colon cancer (Horn et al., 1990).
The signal transduction pathways activated by OSM
result in tyrosine phosphorylation of multiple proteins
(Auguste et al., 1997; Schieven et al., 1992), downregulation of EGF-R-mediated cellular proliferation
(Spence et al., 1997) and a decrease in c-myc expression
(Liu et al., 1997a). The OSM-R itself is also tyrosine
phosphorylated on both the gp 130 and gp 180
subunits (Auguste et al., 1997). However, this is not
autophosphorylation as cytokine receptors do not
contain a catalytic domain (Ihle and Kerr, 1995;
Taniguchi, 1995).
1347
Oncogenic pathways
AC Porter and RR Vaillancourt
1348
Activation of the OSM-R leads to recruitment of the
JAK/STAT (Janus kinase/signal transduction activation of transcription) pathway. JAK is a tyrosine
kinase and has been shown to activate Ras, leading to
Raf, MEK and ultimately MAPK activation in
response to OSM stimulation of HeLa (Stancato et
al., 1997). In addition, OSM is also capable of
activating PI3-K (Ihle and Kerr, 1995). MAPK
activation is responsible for serine phosphorylation of
the STAT proteins which then act as transcription
factors. However, it appears that STAT proteins
require both serine and tyrosine phosphorylation for
activity (Wen et al., 1995). It is activation of the JAK/
STAT pathway which appears to be responsible for the
growth inhibitory e€ects of OSM through changes in
gene transcription (Stancato et al., 1997). It is not
entirely clear what pathway is responsible for the
growth-stimulatory e€ects of OSM but there is some
evidence that the JNK (c-jun N-terminal kinase)
pathway is involved (Liu et al., 1997b). This divergent
signaling by OSM may be the result of cell-type speci®c
expression of signal transduction proteins as well as
OSM receptor subunits.
Recently, cross-talk between RTKs and cytokine
receptors have been observed with the EGF-R and the
growth hormone receptor, respectively. It has been
shown that growth hormone-stimulated activation of
JAK is able to tyrosine phosphorylate the EGF-R
leading to activation of the MAPK signaling pathway,
independent of EGF-R kinase activity and EGF
binding (Yamauchi et al., 1997). Phosphorylation of
the EGF-R results in an interaction between the
receptor and Grb2 which initiates the MAPK
signaling cascade, demonstrating an interaction between cytokine receptors and RTKs. In those
experiments, the cytokines IL-6 and LIF were unable
to stimulate EGF-R phosphorylation, while OSM was
not tested (Yamauchi et al., 1997). However, coadministration of OSM is able to inhibit the
proliferation induced by EGF and basic ®broblast
growth factor (bFGF) in some breast cancer cell lines
(Liu et al., 1997a). This may be due to a dominant
growth-inhibitory signal from the OSM-R, preventing
RTK-dependent signaling for cellular proliferation.
It is interesting to speculate on the functional
signi®cance of this cross-talk between cytokine
receptors and RTKs. Both growth-stimulatory and
growth-inhibitory responses are seen with OSM. In
some cases, this cross-talk may serve as an inhibitory
pathway to impede uncontrolled cellular proliferation.
However, it appears that during the oncogenic process,
constitutively activated RTKs may over-ride the
growth-inhibitory mechanisms of oncostatins or conversely, RTKs may utilize OSM-dependent pathways,
such as the JAK/STAT pathway, as a synergistic
mechanism to promote unregulated cellular proliferation.
Phosphoinositide 3-kinase
Phosphoinositide 3-kinase (PI3-K) can now be added
to the list of proto-oncogenes. PI3-K is a heterodimer
consisting of an 85 kDa regulatory subunit and a 110
kDa catalytic subunit that phosphorylates lipids
(reviewed in Kapeller and Cantley, 1994). Recently,
the catalytic subunit of PI3-K was cloned from the
genome of the avian sarcoma virus 16 (Chang et al.,
1997). The viral gene, termed v-p3k, has a 14 amino
acid deletion at the amino terminus and is fused with
the retroviral gag gene product. In addition, v-p3k
di€ers from c-p3k in that there are four missense
mutations, outside the kinase domain. However, these
mutations do not inhibit the catalytic activity of v-p3k.
In fact, the focus forming activity of v-p3k is ten times
more ecient than c-p3k. These data demonstrate that
PI3-K is an essential component of the cellular
machinery for cellular proliferation.
Many of the RTKs and cytokine receptors that
control cell growth also regulate PI3-K activity.
Mitogenic signaling from the bPDGF-R requires PI3K activity in ®broblasts as determined by site-directed
mutagenesis of tyrosines that are known to associate
with PI3-K (Valius and Kazlauskas, 1993). In MCF-7
human breast cancer cells, IGF-1-dependent mitogenesis requires PI3-K and is independent of MAPK
(Dufourny et al., 1997). PI3-K activity is also required
for interleukin-2 (IL-2)-dependent control of lymphoid
cell growth (Monfar et al., 1995).
The colony-stimulating factor receptor (CSF-1R) is
encoded by the c-fms proto-oncogene and binds
macrophage-CSF. The CSF-1R is structurally related
to the a and b platelet-derived growth factor receptors
(PDGF-R), and the c-ret proto-oncogene product, in
that these receptors have a kinase domain that is
disconnected by a kinase insert domain. CSF-1
regulates macrophage proliferation, di€erentiation,
and survival through the CSF-1R. Of the many signal
transduction pathways that are activated by the CSF1R (reviewed in Hamilton, 1997), the PI3-K pathway
may be important in malignant cell growth. Evidence
for this comes from immunohistochemical studies of
breast tumor specimens which demonstrated that 72%
of CSF-1R positive carcinomas had receptors that were
phosphorylated at Tyr723, which is the PI3-K
recognition site of this receptor (Flick et al., 1997).
Akt
Akt is a proto-oncogene activated by phospholipids
produced PI3-K. The two SH2 domains of the 85 kDa
subunit associate with speci®c phosphotyrosines on
RTKs which e€ectively brings the 110 kDa subunit in
close proximity to the plasma membrane. There, it
catalyzes the phosphorylation of membrane-bound
phosphoinositides at the D-3 hydroxyl of myoinositol, producing the second messengers phosphatidylinositol 3-phosphate [PIP], phosphatidylinositol 3,4bisphosphate [PI(3,4)P2], and phosphatidylinositol
3,4,5-trisphosphate [PI(3,4,5)P3]. The target of the
latter two phospholipids is the proto-oncogene Akt
(Franke et al., 1997; Stokoe et al., 1997)]. Akt is also
known at Protein Kinase B (PKB) and RACPK
(Related to A and C Protein Kinase).
v-Akt was cloned from the genome of the AKT8
virus (Bellacosa et al., 1991). Although the nucleotide
sequence for v-akt has ®ve G?A transitions, the
amino acid sequence is identical to that of c-Akt. The
v-akt oncogene is the product of a recombination
between nucleotides from the viral gag gene, 785
basepair (bp) downstream from the gag ATG codon,
Oncogenic pathways
AC Porter and RR Vaillancourt
and 60 bp upstream of the c-akt ATG codon. An
additional 3 bp were inserted between the gag and cAkt sequences producing a 63 nucleotide insert which,
when translated, retains the c-Akt coding sequence.
Thus, the v-Akt gene product is a hybrid protein
consisting of Gag/63 bp/c-Akt. This gene product was
immunoprecipitated from AKT8-transformed mink
cells, suggesting that v-akt is a cellular proliferation
signal (Bellacosa et al., 1991). As further evidence to
implicate v-akt in malignant cell growth, Akt2, a
homologue of Akt, is over-expressed in some human
ovarian carcinoma cell lines and primary tumors
(Cheng et al., 1992).
c-Akt consists of 480 amino acids where amino acids
1 ± 147 compose the AH (Akt homology) domain and
amino acids 1 ± 106 are referred to as the PH
(pleckstrin homology) domain. The AH and PH
domains are involved in protein-protein interactions
(Datta et al., 1995) as well as the interaction with
phospholipids. The membrane-bound phospholipids,
PI(3,4)P2 and PI(3,4,5)P3, bind to the pleckstrin
homology (PH) domain of Akt (Figure 1), thus
recruiting Akt to the plasma membrane and activating
its kinase activity (Franke et al., 1997; Klippel et al.,
1997; Stokoe et al., 1997). When Akt binds these
inositol phosphates, it becomes phosphorylated, by an
unknown kinase, and Akt protein kinase activity
increases (Stokoe et al., 1997). Thus, Akt is the target
of phospholipid second messengers.
PDGF, EGF and bFGF are potent activators of
PI3-K and activation of their respective receptors
increases Akt kinase activity (Burgering and Co€er,
1995; Franke et al., 1995). Thus, PI3-K functions to
link growth factor receptors with Akt. So then, what
does Akt phosphorylate? Recently, the apoptotic/antiapoptotic protein, BAD, has been identi®ed as an
Akt substrate where phosphorylation of BAD is
regulated by the growth factors PDGF and IGF-1
(Datta et al., 1997), as well as the cytokines, IL-2
(Ahmed et al., 1997) and IL-3 (del Peso et al., 1997).
The signi®cance of these ®ndings is that when
phosphorylated, BAD functions as an anti-apoptotic
protein, promoting cell survival (Zha et al., 1996).
Thus, growth factors and cytokines mediate proliferative as well as anti-apoptotic signals. At this time,
it has not been determined whether v-Akt phosphorylates BAD. However, it is tempting to speculate that
BAD will be a substrate for v-Akt. As a universal
mechanism, maintenance of cell survival by phosphorylation of BAD would serve to insure the
transforming potential of oncogenes.
Glycogen synthase kinase 3 (GSK3) is another
substrate for Akt phosphorylation. Serine phosphorylation of GSK3 results in inactivation of GSK3
(Sutherland et al., 1993) which may disrupt the
degradation pathway for free b-catenin in the cell.
This result is signi®cant because excess b-catenin has
been shown to play a role in tumorigenesis due to its
transcriptional activity (Rubinfeld et al., 1997).
b-catenins
RTKs may also transduce oncogenic signals through bcatenins (Figure 1). Evidence suggests that there are
two di€erent roles played by b-catenins in the cell. One
role casts b-catenins as structural components of the
cell adhesion/actin cytoskeleton network (Daniel and
Reynolds, 1997). Another role casts free b-catenins as
signaling molecules involved in gene transcription
(Korinek et al., 1997). Both of these functions may
contribute to b-catenin involvement in the development
of cancer. The loss of cell/cell adhesion, mediated by bcatenins, may be a factor in anchorage-independent cell
growth and cancer metastasis. In addition, gene
transcription mediated by b-catenins may turn on
genes required for cellular proliferation or genes with
an anti-apoptotic function (Peifer, 1997). RTKs may be
directly or indirectly involved in both functions of bcatenins.
Cadherins are transmembrane glycoproteins which
provide an anchor through cell-cell contact. Intracellularly, cadherins interact directly with b-catenins which
provide a link to the actin cytoskeleton through an
interaction with a-catenin. Loss of this link is thought
to be involved in the development of metastatic cancers
(reviewed in Daniel and Reynolds, 1997). Activation of
RTKs, particularly by EGF and HGF/SF, has been
shown to induce scattering of human cancer cells via
tyrosine phosphorylation of b-catenin (Shibamoto et
al., 1994). Whether the phosphorylation is mediated by
the RTK, or through activation of a soluble protein
tyrosine kinase by the RTK, is not yet clear. However,
a direct interaction has been shown between the EGFR and b-catenin in vitro and EGF induces immediate
b-catenin phosphorylation (Hoschuetzky et al., 1994).
It is possible that phosphorylation of b-catenin severs
its interaction with the actin cytoskeleton (Daniel and
Reynolds, 1997). It is also possible that phosphorylation of b-catenin increases the pool of free b-catenin in
the cell. This may be the link between activation of
RTKs and cellular growth, that is characterized by the
loss of contact inhibition, which is common in
metastatic tumor formation.
In addition to its role in cell-cell interactions, free bcatenin plays a role in cellular signal transduction that
has only recently been identi®ed (Peifer, 1997). Free bcatenin is capable of forming a complex with DNA
binding proteins of the T cell factor (Tcf) and
lymphoid enhancer factor (Lef) families. Tcf and Lef
interact with b-catenin and the subsequent regulation
of gene transcription is thought to play an important
role in tumorigenesis by activating genes involved in
proliferation and/or genes with anti-apoptotic functions.
The levels of free b-catenin, as well as its stability,
can be increased in some forms of cancer, leading to
unregulated gene transcription. Some colon cancer cell
lines are marked by the lack of tumor suppressor,
adenomatous polyposis coli (APC), which leads to the
constitutive activation of b-catenin-mediated signal
transduction (Korinek et al., 1997). In addition,
deletions, truncations, and point mutations have been
found in both APC and b-catenin in colon cancer cell
lines (Morin et al., 1997). The mutations in APC
disrupt interactions with b-catenin and lead to
constitutive transcriptional activity of b-catenin.
Deletions or point mutations in b-catenin either
remove serines or change serines to other amino
acids. Interestingly, these serines are potential phosphorylation sites for GSK3 (Morin et al., 1997). As a
functional consequence, loss of serines in b-catenin
1349
Oncogenic pathways
AC Porter and RR Vaillancourt
1350
leads to increased transcriptional activity and insensitivity to APC-mediated down-regulation of b-catenin.
In melanoma cell lines, very high levels of free bcatenin are often present. As in colon cancer cell lines,
point mutations resulting in the loss of critical serines
or truncations of the amino terminal region of bcatenin have been detected. In addition, mutations and
loss of APC were found in melanoma cell lines. A
single point mutation, S37?F, appears to be
responsible for the stabilization of b-catenin and a
greater than tenfold increase in the half life of the
protein in the cell (Rubinfeld et al., 1997). The loss of
serines reduced phosphorylation of b-catenin, producing higher levels of free b-catenin which is capable of
regulating transcriptional activity.
RTKs regulate the activity of b-catenin by two
mechanisms. First RTKs may be responsible for direct
phosphorylation of b-catenin which results in a loss of
contact with the actin cytoskeleton and ultimately a
loss of cell/cell adhesion (Daniel and Reynolds, 1997).
This may contribute to cancer metastasis. Secondly,
RTKs may regulate the transcriptional activity of bcatenin via a PI3-K dependent pathway.
RTKs activate PI3-K to produce phospholipid
second messengers which are involved in activation of
Akt (Franke et al., 1997). Akt then phosphorylates and
inhibits the activity of GSK3. GSK3b has been shown
to phosphorylate APC (Rubinfeld et al., 1996) and bcatenin (Peifer et al., 1994). Inhibition of GSK3
activity prevents phosphorylation of APC. Formation
of a complex between b-catenin, APC, and GSK3 is
dependent upon phosphorylation of APC (Rubinfeld et
al., 1996). It is the formation of this complex which is
responsible for degradation of excess free b-catenin.
Thus, activation of RTKs disrupts degradation of bcatenin. This pool of free b-catenin is then able to
function as a transcriptional activator and turn on
genes involved in proliferation and/or inhibition of
apoptosis.
Because the GSK3/APC/b-catenin complex is not
formed due to inactivation of GSK3 in RTK
stimulated cells, APC may then be capable of playing
a role in cell migration and motility. Nathke et al.,
(1996) have shown that APC localizes to the ends of
microtubules in actively migrating areas of epithelial
cell membranes. This localization and stimulation with
HGF indicate that APC plays an active role in cell
migration. In addition, APC is involved in normal cell
migration from the crypt in the intestine. Together
these data indicate that APC is a key component of cell
migration. Then it is possible that, in RTK stimulated
cells in which GSK3 is inactivated by Akt phosphorylation, APC may be involved in aberrant migratory
activity associated with tumor progression.
of the PDGF B chain, is an oncogene. Inappropriate
expression of v-sis e€ectively activates the PDGF-R to
produce a persistent mitogenic signal in cells.
The inappropriate expression of a growth factor can
function as an oncogenic mechanism to induce cell
growth or cell survival. Aside from promoting cell
proliferation and di€erentiation, oncogenic Ras can
also induce over-expression of the growth factor IGF-1
(Dawson et al., 1995). One of the major functions of
IGF-1 is to function as a survival factor in serum,
protecting cells from apoptosis. The IGF-1R activates
PI3-K whose phospholipid products bind to the PH
domain of Akt, producing an anti-apoptotic signal. In
the cascade of signals, Akt binds PI3-K-generated
phospholipids which activate Akt to phosphorylate
BAD, which is an anti-apoptotic signal in an IL-3dependent hematopoietic cell line (Zha et al., 1996).
Therefore, activated Ras may function to e€ect
autocrine loops that produce growth factors, such as
IGF-1, whose function is to maintain cell survival by
inhibiting apoptosis, thus allowing mutated cells to
proliferate (Figure 1).
Concluding remarks
In conclusion, it is not only RTKs and their growth
factor ligands that function as oncogenes. Many of the
proteins involved in RTK signal transduction are also
capable of acting as oncogenes, independent of RTK
stimulation. RTKs have their e€ect on cellular
physiology via a number of di€erent mechanisms,
including increased proliferation, disruption of apoptosis, disruption of normal cytoskeletal interactions,
increased cell motility, and invasive cell growth. RTK
and Ras-mediated production of growth factors can
activate a number of these pathways due to autocrine
and paracrine loops. In addition, activation of PI3-K
by oncogenic RTKs can lead to changes in gene
transcription, cell motility, cytoskeletal changes, and
anti-apoptotic signals; all of which could contribute to
oncogenesis. Moreover, there is evidence that there
may be cross-talk between the RTKs and the
oncostatin cytokine receptor which could in¯uence
proliferation. The picture of RTK signal transduction
which leads to oncogenesis is far from complete.
However, as we learn more about oncogenes and
signal transduction pathways, we are beginning to
appreciate that oncogenes utilize multiple pathways for
their selective growth advantages. De®ning these
pathways and mechanisms will ultimately lead to a
better understanding of the oncogenic process, which
may provide more selective methods for the treatment
of cancers.
Autocrine/paracrine loops
In addition to receptor tyrosine kinases, growth factors
may also function as oncogenes. Retroviruses contain
within their genome oncogenic forms of growth
factors. For example, although the PDGF receptors
are not proto-oncogenes, v-sis, which is a homologue
Acknowledgements
The authors are supported, in part, by grants from the
American Cancer Society (IRG 110T), the Southwest
Environmental Health Sciences Center (ES 06694), and a
grant from the Arizona Disease Control Research Commission.
Oncogenic pathways
AC Porter and RR Vaillancourt
1351
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